(Amido)N-substituted bleomycins, salts thereof and process for preparation thereof

ABSTRACT

A promising carcinostatic (amido)N-substituted bleomycin represented by the following formula, or a salt thereof and a process for the preparation thereof: ##STR1## wherein BM represents a moiety of bleomycin skeleton; X represents an alkyl of 1 to 18 carbon atoms, an aminoalkyl of 1 to 12 carbon atoms, a lower alkyl having a halogen, phenyl indolyl, 5- or 6-membered heterocyclic group, or (lower)alkylamino (the alkyl group may have a substituent group) as a substituent, naphthyl, thiazolyl, or N-phenyl(lower)alkylpiperazinyl; and R represents a terminal amino residue of the bleomycin.

This invention relates to novel (amido)N-substituted bleomycins, saltsthereof and a process for the preparation thereof.

Bleomycin is a family of carcinostatic antibiotic substances discoveredin 1966 by Umezawa, one of the present inventors, and collaborators[Journal of Antibiotics, 19A, p. 200 (1966)]. It is produced byStreptomyces verticillus, an Actinomycete, and is a basic water-solubleglycopeptide capable of readily chelating one atom of divalent copper.In ordinary culture, 16 members of the bleomycin family are produced andare each isolated [e.g., Umezawa et al., Journal of Antibiotics, 19A, p.210 (1966)]. Of these bleomycins, A₁, A₂, A₅, B₂ and demethyl-A₂ arecurrently being widely used in the form of copper-free mixture(hereinafter referred to as bleomycin complex) in clinical fields ofcancer therapy; more particularly, they are successfully used in thetreatment of squamous cell carcinoma as major target, skin cancer, headand neck cancer, lung cancer, and malignant lymphoma. Various bleomycinsare also disclosed in U.S. Pat. No. 3,922,262 and U.S. Pat. No. Re30,451.

The bleomycins are generally produced in copper-containing form in theordinary fermentation. The copper-free form is obtained by removing thecopper from the copper-containing form. The term "bleomycin", as hereinemployed, includes both the copper-containing and the copper-free forms,unless specifically indicated.

The bleomycins are represented by the general formula (II) ##STR2##wherein R is a terminal amine residue of bleomycin; and the chelatedcopper is omitted in the case of copper-containing form.

It was found, however, that the bleomycins are inactivated by the actionof a bleomycin-inactivating enzyme [hereinafter referred to briefly asinactivating enzyme; Umezawa et al., Journal of Antibiotics, Vol. 27, p.419 (1974)]. It was further found that the bleomycins are relativelyless inactivated in the skin and lung where they exhibit a highactivity, while they are easily inactivated in the stomach where theyhave been believed to exhibit no action and that such an inactivationphenomenon is less marked in the squamous cell carcinoma in mouse thanin the sarcoma in mouse, both of which are induced by20-methylcholanthrene [Umezawa et al., journal of Antibiotics, Vol. 25,p. 409 (1972); Vol. 27, p. 419 (1974)]. Furthermore, it was found thatthe bleomycin-inactivating action is exhibited by the squamous cellcarcinoma in human head and neck, especially by those of the lowdifferentiation type against which the bleomycins have been believed tobe not so effective [Mueller et al., Cancer, Vol. 40, p. 2787 (1977)].

As is understandable from the above reports, the bleomycins areincapable of exhibiting a sufficient activity against carcinomascontaining bleomycin-inactivating enzyme of high activity. This is oneof the reasons for the need of further improvement in bleomycins. Thepresent inventors contemplated that if it is possible to discover ableomycin derivative difficulty susceptible to enzymatic inactivation,it would become possible to treat more effectively, for example, headand neck cancer, esophagus cancer, lung cancer, and squamous cellcarcinomas in other regions and to treat those adenocarcinomas such asgastric cancer which are not responsive to the treatment withconventional bleomycins. From such a viewpoint the present inventorsmade an extensive study and, as a result, found that the bleomycins areinactivated to a lesser degree when a substituent (-X) is introduced tothe nitrogen atom in amide linkage of the partial structure,2,3-diaminopropanamide ##STR3## of bleomycins. The present invention isbased on this finding.

The primary object of this invention is to provide novel bleomycinswhich are difficulty inactivated by the inactivating enzyme and toprovide a process for the preparation thereof.

Other objects and advantages of this invention will become apparent fromthe following description.

According to this invention there are provided (amido)N-substitutedbleomycins or salts thereof, intermediates for the preparation thereof,and a process for the preparation thereof, said (amido)N-substitutedbleomycins being represented by the general formula ##STR4## wherein BMrepresents a moiety of bleomycin skeleton; X represents (1) an alkyl of1 to 18 carbon atoms, (2) an aminoalkyl of 1 to 12 carbon atoms, (3) alower alkyl having as substitutent (a) 1 to 3 halogen atoms, (b) 1 or 2phenyl groups, (c) an indolyl group, or (d) a 5- or 6-memberedheterocyclic group containing an oxygen, sulfur or nitrogen atom (amongthe substituent groups, the phenyl or indolyl group may be furthersubstituted by a halogen atom or a lower alkoxy group), (4) X₁-(lower)alkyl [where X₁ is ##STR5## X₂ is a hydrogen atom, a lower alkylor benzyl, X₃ is (a) a lower alkyl, (b) a phenyl(lower)alkyl, or (c) amono- or di-(lower)alkylamino(lower)alkyl which may be substituted by aphenyl or halophenyl group, X₄ is (a) a lower alkyl or (b) aphenyl(lower)alkyl] (5) naphthyl, (6) thiazolyl, or (7) anN-phenyl(lower)alkylpiperazinyl; and R represents a terminal aminoresidue of the bleomycin.

The moiety of bleomycin skeleton represented by BM is the portion of ableomycin molecule enclosed in dotted lines in the general formula (II)of bleomycins and includes both the copper-containing form and thecopper-free form, unless specifically indicated. The lower alkyls arealkyls of 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, n-penthyl, 3-methylbutyl, 3-methylpentyl,and n-hexyl.

Examples of individual groups represented by X in the general formula(I) are as follows:

(1) Alkyls of 1 to 18 carbon atoms: methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, tert-butyl, n-pentyl, 3-methylbutyl, neopentyl,n-heptyl, 3-methylpentyl, n-hexyl, 1,5-dimethylhexyl, isohexyl, n-octyl,n-decyl, lauryl, myristyl, cetyl, and stearyl.

(2) Aminoalkyls of 2 to 12 carbon atoms: 2-aminoethyl, 3-aminopropyl,4-aminobutyl, 6-aminohexyl, 12-aminododecyl, and4-amino-4-methyl-1-dimethylpentyl.

(3) Lower alkyls having as substituent (a) 1 to 3 halogen atoms, (b) 1or 2 phenyl groups, (c) an indolyl group, or (d) a 5- or 6-memberedheterocyclic group containing an oxygen, sulfur or nitrogen atom (amongthe substituent groups, the phenyl or indolyl group may be furthersubstituted by a halogen atom or a lower alkoxy group):2,2,2-trifluoroethyl, 2,2,2-trichloroethyl, 2,2-difluoroethyl,2,2-dichloroethyl, benzyl, diphenylmethyl, 2-phenylethyl,2,2-diphenylethyl, 1-phenylethyl, 1,2-diphenylethyl, 3-phenylpropyl,2-phenylisopropyl, 1,3-diphenylpropyl, 3,3-diphenylpropyl,4-phenylbutyl, 4,4-diphenylbutyl, chlorobenzyl, dichlorobenzyl,bromobenzyl, methoxybenzyl, dimethoxybenzyl, ethoxybenzyl,methylenedioxybenzyl, propoxybenzyl, p-chlorophenylethyl,p-methoxyphenylethyl, p-benzyloxybenzyl, furylmethyl, 2-furylethyl,2-thiazolylmethyl, 2-pyrazolylmethyl, 2-imidazolylmethyl,4-imidazolylmethyl, 2-thienylmethyl, 2-pyridylmethyl, 3-pyridylmethyl,4-pyridylmethyl, 3-indolylmethyl, 2-(3-indolyl)ethyl,4-methoxy-3-indolylmethyl, 2-pyrimidylmethyl, 2-pyrimidylethyl,4-pyrimidylmethyl, 4-pyrimidylethyl, 2-piperidylmethyl,3-piperidylmethyl, 4-piperidylmethyl, 2-(2-piperidyl)ethyl,2-(3-piperidyl)ethyl, 2-(4-piperidyl)ethyl, 1-(2-piperidyl)ethyl,1-(3-piperidyl)ethyl, 1-(4-piperidyl)ethyl, 2-(piperidino)ethyl,2-piperazylmethyl, 2-(2-piperazylethyl), 2-(piperidino)ethyl,3-piperidinopropyl, 2-(morpholino)ethyl, 3-(morphilino)propyl,2-morpholinylmethyl, 3-morpholinylmethyl, 2-(morpholinyl)ethyl, and3-(morpholinyl)propyl.

(4) X₁ -(lower)alkyls (where X₁ is ##STR6## dimethylaminoehtyl,diethylaminoethyl, dipropylaminoethyl, propylaminoethyl,dimethylaminopropyl, diethylaminopropyl, dipropylaminopropyl,propylaminopropyl, dibutylaminopropyl, butylaminopropyl,benzylaminopropyl, 2-phenylethylaminopropyl, 1-phenylethylaminopropyl,3-phenylpropylaminopropyl, 4-phenylbutylaminopropyl,methylaminoethylaminopropyl, ethylaminopropylaminopropyl,propylaminopropylaminopropyl, butylaminopropylaminopropyl,N-(butylaminopropyl)-N-methylaminopropyl, dibutylaminopropylaminopropyl,penthylaminopropylaminopropyl, ethylaminobutylaminopropyl,propylaminobutylaminopropyl, ethylaminopropylaminobutyl,butylaminopropylaminobutyl, benzylaminoethyl aminoethyl,benzylaminoethylaminopropyl, benzylaminopropylaminopropyl,benzylaminobutylaminopropyl, phenylethylaminopropylaminopropyl,N-(phenylethylaminopropyl)-N-methylaminopropyl,N-(chlorobenzylaminopropyl)-N-methylpropyl,N-(bromobenzylaminopropyl)-N-methylpropyl,N-(chlorophenylethylaminopropyl)-N-methylpropyl,benzylaminopropyl-N,N-dimethylaminopropyl,dibenzylaminopropyl-N,N-diethylaminopropyl, anddibenzylaminopropyl-N-methyl-N-benzylaminopropyl.

(5) Naphthyls: α-naphthyl and β-naphthyl.

(6) Thiazolyls: 2-thiazolyl, 3-thiazolyl, and 4-thiazolyl.

(7) N-Phenyl(lower)alkylpiperidyls: N-benzyl-4-piperidyl,N-phenylethyl-4-piperidyl, N-benzyl-3-piperidyl, andN-benzyl-2-piperidyl.

The terminal amino residue of bleomycins represented by R may be any ofthe substituted or unsubstituted aliphatic amino groups, but it isusually an aliphatic primary amino group of basic nature represented bythe general formula (III)

    R.sub.2 --R.sub.1 --NH--                                   (III)

wherein R₁ is (1) a chain of alkylene groups which may have a nitrogenatom intervened between alkylene groups in the chain [examples of such agroup are those represented by the formulas --R₁₂ --Y₁ --R₁₃ -- and--R₁₂ --Y₁ --R₁₃ --Y₂ --R₁₄ --, wherein R₁₂, R₁₃ and R₁₄ are alkylenegroups and Y₁ and Y₂ are each a group of the formula ##STR7## (where R₃and R₄ are hydrogen atoms or lower alkyls which may have substituents)],(2) ##STR8## or (3) phenylene; and R₂ is any of the groups of basicnature [examples of such a group are those represented by the formula##STR9## wherein R₄ is as defined above; R₅, R₆ and R₉ are each a loweralkyl which may have a substituent; and R₇ and R₈ are each a hydrogenatom or an alkyl of 1 to 10 carbon atoms which may have a substituent].The alkylene of the above amino group is that of 1 to 8 carbon atomssuch as, for example, --CH₂ --, --(CH₂)₂ --, ##STR10## --(CH₂)₃ --,##STR11## --(CH₂)₄ --, --(CH₂)₅ --, --(CH₂)₆ --, and --(CH₂)₈ --, thoughthe alkylenes having 2 to 4 carbon atoms are most frequently used. Thesubstituents on the above alkyl groups include hydroxyl, alkoxy (forexample, methoxy, ethoxy, propoxy and butoxy), phenyl [which may haveone or more substituents selected from halogens, cyano, lower alkyls,benzyloxy, and substituted (e.g. alkoxy-, phenoxy-, orhalogen-substituted) benzyloxy groups], and cycloalkyls of 5 to 13carbon atoms.

Examples of the above amino groups are 2-aminoethylamino,3-aminopropylamino, 2-dimethylaminoethylamino, 2-diethylaminoethylamino,3-dimethylaminopropylamino, 3-diethylaminopropylamino,3-(3-butylaminopropylamino)propylamino, 3-(2-oxypropylamino)propylamino,3-piperidinopropylamino, 3-(1-phenylethylamino)propylamino,2-aminopropylamino, 3-methylaminopropylamino, 3-butylaminopropylamino,3-(6-aminohexylamino)propylamino, 3-trimethylaminopropylamino,3-(3-dimethylaminopropylamino)propylamino,3-(3-aminopropylamino)propylamino,3-[N-methyl-N-(3-aminopropyl)amino]propylamino,3-pyrrolidinylpropylamino, 3-piperidinylpropylamino,3-morpholinopropylamino, 3-piperazinylpropylamino,3-[4-(3-aminopropylpiperazinyl)]propylamino,3-(3-pyrrolidinylpropylamino)propylamino,3-(3-piperdinylpropylamino)propylamino,3-(3-morpholinylpropylamino)propylamino,3-(3-oxypropylamino)propylamino, 3-(3-methoxypropylamino)propylamino,3-benzylaminopropylamino, m-aminomethylbenzylamino,p-aminomethylbenzylamino, 2-cyclopentylaminoethylamino,3-cyclohexylaminopropylamino, 4-cyclohexylaminobutylamino,cycloheptylaminopropylamino, 3-cyclooctylaminopropylamino,3-[N-methyl-N-(3-cyclooctyl methylaminopropyl)amino]propylamino,3-cyclodecanylmethylaminopropylamino,3-{N-methyl-N-[3-(2-p-chlorophenylethylamino)propyl]amino}propylamino,3-{N-methyl-N-[3-(m,p-dibenzyloxybenzyl)aminopropyl]amino}propylamino,3-{N-methyl-N-[3-(p-cyanobenzylamino)propyl]amino}propylamino,3-{N-methyl-N-[3-(cycloundecanylmethylamino)propyl]amino}propylamino,3-{N-methyl-N-[bis(m,p-dibenzyloxybenzyl)aminopropyl]amino}propylamino,3-{N,N-dimethyl-N-[3-(dibenzylamino)propyl]amino}propylamino,3-{N,N-diethyl-N-[3-(dibenzylaminopropyl)]amino}propylamino,3-{N,N-dimethyl-N-[3-(N,N-dimethyl-N-(3-dibenzylaminopropyl)amino)propyl]amino}propylamino,3-{N,N-dimethyl-N-[3-(N,N-dimethyl-N-(3-cyclooctylmethylaminopropyl)amino)propyl]amino}propylamino,3-[4-(3-dibenzylaminopropyl)piperidyl]propylamino, and3-{4-[3-(cyclooctylmethylamino)propyl]piperidyl}propylamino.

As desirable compounds, mention may bemade of those in which R is3-[(S)-1'-phenylethyl]aminopropylamino or3'-(n-butylaminopropyl)aminopropylamino and those having an amineresidue represented by the formula (IV)

    --NH--(CH.sub.2).sub.3 --A--(CH.sub.2).sub.3 --B           (IV)

wherein A is ##STR12## (where R₁₀, R₁₀ ', R₁₁ and R₁₁ ', which may bethe same or different, are each a lower alkyl, benzyl, or ahalogen-substituted benzyl) and B is ##STR13## [where R₁₂ is (1) aphenyl(lower)alkyl, (2) a phenyl(lower)alkyl having on the phenylnucleus one or more substitutents selected from (a) halogens, (b) loweralkyls, (c) lower alkoxys, (d) cyano, (e) trifluoromethyl, (f)benzlyloxy which may have on the phenyl nucleus such substitutents ashalogens, alkoxy and phenoxy, (g) di(lower)alkylamino groups, and (h)phenyl, (3) cyclohexyl, (4) triphenylmethyl, (5) naphthylmethyl, (6)furylmethyl, (7) thiophenemethyl, (8) a lower alkyl substituted by acycloalkyl of 5 to 13 carbon atoms, or (9) norbornene-2-methyl; and R₁₃is (1) hydrogen, (2) benzyl, or (3) benzyl having on the phenyl nucleusone or more halogens or benzyloxy groups are substitutent]. Thesecompounds are desirable because of their relatively low pulmonarytoxicity. In the above formulas, lower alkyls include methyl, ethyl, andbutyl; phenyl(lower)alkyls include benzyl, 2-phenylethyl, 1-phenylethyl,3-phenylpropyl, 2,2-diphenylethyl, and dibenzylmethyl;phenyl(lower)alkyls having substituents on the phenyl nuclei include2-p-chlorophenylethyl, p-chlorobenzyl, o-chlorobenzyl, m-chlorobenzyl,1-(p-chlorophenyl)ethyl, o,p-dichlorobenzyl, m,p-dichlorobenzyl,p-bromobenzyl, p-fluorobenzyl, pentafluorobenzyl,m-trifluoromethylbenzyl, p-methylbenzyl, p-diethylaminobenzyl,p-methoxybenzyl, o,p-dimethoxybenzyl, m,p-dibenzyloxybenzyl,p-cyanobenzyl, and p-phenylbenzyl; and lower alkyls substituted by acycloalkyl of 5 to 11 carbon atoms include cyclopentylmethyl,cyclohexylmethyl, cycloheptylmethyl, cyclooctylmethyl,cycloundecanylmethyl, and 2-cyclohexylethyl.

Especially desirable compounds are those represented by the generalformula (I) in which R is 1-phenylethylaminopropylamino,butylaminopropylamino, or an amino group represented by the generalformula (V)

    --NH--(CH.sub.2).sub.3 --A.sub.1 --(CH.sub.2).sub.3 --B.sub.1 (V)

wherein A₁ is ##STR14## (where R₁₀ ' and R₁₁ ' are each a lower alkyl orbenzyl) and B₁ is ##STR15## [where R₁₂ ' is (1) a phenyl(lower)alkylwhich may have on the phenyl nuclues one or two substituents including(a) halogens, (b) cyano, and (c) benzyloxy group, or (2) a lower alkylsubstituted by a cycloalkyl of 5 to 13 carbon atoms; and R₁₃ ' ishydrogen or benzyl which may have one or two benzyloxy groups assubstituents].

The combinations of X and R, which show an especially low pulmonarytoxicity, are as follows:

(Amido)N-substituted bleomycins in which X is isopropyl,(S)-1-phenylethyl, diethylamino-1-methylbutyl, dibutylaminopropyl, orn-butylaminopropylamino and R is 1-phenylethylamino orN-(lower)alkyl-N-(halobenzylaminopropyl)aminopropylamino; salts thereof;(amido)N-substituted bleomycins in which X is an alkyl of 3 to 8 carbonatoms, 1,5-dimethylhexyl, benzyl, 1-phenylethylaminopropyl,di-n-butylaminopropyl, n-butylaminopropylaminopropyl,N-methyl-N-benzyl-N-(dibenzylaminopropyl)aminopropyl, orN-methyl-N-(halophenylethylaminopropyl)aminopropyl and R isN-methyl-N-(halophenylethylaminopropyl)aminopropylamino,N-methyl-N-benzyl-N-(dibenzylaminopropyl)aminopropylamino,N-methyl-N-[bis(m,p-dibenzyloxybenzyl)aminopropyl]aminopropylamino,N-methyl-N-methy-N-(dibenzylaminopropyl)aminopropylamino,N-ethyl-N-ethyl-N-(dibenzylaminopropyl)aminopropylamino,N-methyl-N-(cyclooctylmethylaminopropyl)aminopropylamino, orN-metyl-N-(cyanobenzylaminopropyl)aminopropylamino; and salts thereof.

As examples of the compounds of this invention, mention may be made ofthose shown in Table 1. In Table 1, "BLM" in the name of compound standsfor "bleomycin" and the compound represented by the general formula (I)is designated as "name of R (amino residue)-(amido)N-[name of X]-BLM".

                                      TABLE 1                                     __________________________________________________________________________    Compound                                                                      No.   Name of compound                 Abbreviation                           __________________________________________________________________________    1     2-(4'-Imidazolyl)ethylamino-(amido)N--[methyl]-BLM                                                             dH-MMA                                 2     2-(4'-Imidazolyl)ethylamino-(amido)N--[ethyl]-BLM                                                              dH-MEA                                 3     2-(4'-Imidazolyl)ethylamino-(amido)N--[isopropyl]-BLM                                                          dH-IPA                                 4     2-(4'-Imidazolyl)ethylamino-(amido)N--[benzyl]-BLM                                                             dH-BA                                  5     2-(4'-Imidazolyl)ethylamino-(amido)N--[3-((S)-1'-phenylethyl)-                                                 dH-PEP                                       aminopropyl]-BLM                                                        6     2-(4'-Imidazolyl)ethylamino-(amido)N--[2-aminoethyl]-BLM                                                       dH-EDA                                 7     4-Guanidinobutylamino-(amido)N--[isopropyl]-BLM                                                                dB2-IPA                                8     4-Guanidinobutylamino-(amido)N--[1',6'-dimethylheptyl]-BLM                                                     dB2-DHA                                9     4-Guanidinobutylamino-(amido)N--[2',2',2'-trifluoroethyl]-BLM                                                  dB2-TFEA                               10    4-Guanidinobutylamino-(amido)N--[octyl]-BLM                                                                    dB2-OCT                                11    4-Guanidinobutylamino-(amido)N--[3-((S)-1'-phenylethyl)amino-                                                  dB2-PEP                                      propyl]-BLM                                                             12    4-Guanidinobutylamino-(amido)N--[3-di-n-butylamino)propyl]-BLM                                                 bB2-BPA                                13    4-Guanidinobutylamino-(amido)N--[lauryl]-BLM                                                                   dB2-LAA                                14    4-Guanidinobutylamino-(amido)N--[benzyl]-BLM                                                                   dB2-BA                                 15    4-Guanidinobutylamino-(amido)N--[diphenylmethyl]-BLM                                                           dB2-ADPM                               16    4-Guanidinobutylamino-(amido)N--[1',2'-diphenylethyl]-BLM                                                      dB2-DPE                                17    4-Guanidinobutylamino-(amido)N--[β-naphthyl]-BLM                                                          dB2-NA                                 18    4-Guanidinobutylamino-(amido)N--[2-furylmethyl]-BLM                                                            dB2-FFA                                19    4-Guanidinobutylamino-(amido)N--[3-pyridylmethyl]-BLM                                                          dB2-AMPY                               20    4-Guanidinobutylamino-(amido)N--[2-thiazolylmethyl]-BLM                                                        dB2-ATZ                                21    3-((S)-1'-phenylethyl)aminopropylamino-(amido)N--[isopropyl]-BLM                                               dPEP-IPA                               22    3-((S)-1'-phenylethyl)aminopropylamino-(amido)N--[3-((S)-1'-                                                   dPEP-PEP                                     phenylethyl)aminopropyl]-BLM                                            23    3-((S)-1'-phenylethyl)aminopropylamino-(amido)N--[benzyl]-BLM                                                  dPEP-BA                                24    3-((S)-1'-phenylethyl)aminopropylamino-(amido)N--[p-chlorobenzyl]-                                             dPEP-CBA                                     BLM                                                                     25    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[stearyl]-BLM                                                 dPEP-STE                               26    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[lauryl]-BLM                                                  dPEP-LAA                               27    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[octyl]-BLM                                                   dPEP-OCT                               28    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[5-methyoxyindol-                                             dPEP-MTA                                     3-ylethyl]-BLM                                                          29    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[                                                             dPEP-DADdo-                                  decanyl]-BLM                                                            30    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[1',2'-diphenyl-                                              dPEP-DPE                                     ethyl]-BLM                                                              31    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[diphenylmethyl]-                                             dPEP-ADPM                                    BLM                                                                     32    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[1,6-dimethyl-                                                dPEP-DHA                                     heptyl]-BLM                                                             33    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[2-(1-                                                        dPEP-APZ                                     piperazinyl)ethyl]-BLM                                                  34    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[3-(di-n-                                                     dPEP-BPA                                     butylamino)propyl]-BLM                                                  35    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[N--benzyl-                                                   dPEP-ABP                                     piperazin-4-yl]-BLM                                                     36    3-((S)-1'-phenylethylamino)propylamino-(amido)N--[4-diethylamino-                                              dPEP-ADP                                     1-methylbutyl]-BLM                                                      37    3-[N--methyl-N--(3-n-butylaminopropyl)amino]propylamino-                                                       dBAPP-IPA                                    (amido)N--[isopropyl]-BLM                                               38    3-[N--methyl-N--(3-n-butylaminopropyl)amino]propylamino-                                                       dBAPP-BAPP                                   (amido)N--{3-[N--methyl-N--(3-n-butylaminopropyl)amino]-                      propyl}-BLM                                                             39    3-{N--methyl-N--[3-(p-chlorobenzyl)aminopropyl]amino}-                                                         dMCLBZ-PEP                                   propylamino-(amino)N--[3-(S)-1'-phenylethyl)aminopropyl]-BLM            40    3-{N--methyl-N--[3-(p-chlorobenzyl)aminopropyl]amino}-                                                         dMCLBZ-OCT                                   propylamino-(amido)N--[octyl]-BLM                                       41    3-{N--methyl-N--[3-(p-chlorobenzyl)aminopropyl]amino}propyl-                                                   dMCLBZ-                                      amino-(amido)N--[ 1,2-diphenylethyl]-BLM                                                                       DPE                                    42    3-{N--methyl-N--[3-(p-chlorobenzyl)aminopropyl]amino}propyl-                                                   dMCLBZ-                                      amino-(amido)N--[1,5-dimethylhexyl]-BLM                                                                        DHA                                    43    3-{N--methyl-N--[3-(2-(p-chlorophenyl)ethyl)aminopropyl]amino}-                                                dMCLPE-                                      propylamino-(amido)N--[3-((S)-1'-phenylethyl)aminopropyl]-BLM                                                  PEP                                    44    3-{N--methyl-N--[3-(2-p-chlorobenzyl)ethyl)aminopropyl]amino}-                                                 dMCLPE-                                      propylamino-(amido)N--[octyl]-BLM                                                                              OCT                                    45    3-{N--methyl-N--[3-(2-(p-chlorophenyl)ethyl)aminopropyl]amino}-                                                dMCLPE                                       propylamino(amido)N--{3-[N-- methyl-N--[3-(2-(p-chlorophenyl)-                                                 MCLPE                                        ethyl)aminopropyl]amino]propyl}-BLM                                     46    3-{N--methyl-N--[3-(2-(p-chlorophenyl)ethyl)aminopropyl]amino}-                                                dMCLPE                                       propylamino-(amido)N--[3-(di-n-butylamino)propyl]-BLM                                                          BPA                                    47    3-{N--methyl-N--benzyl-N--[3-(dibenzylamino)propylamino}-                                                      dMTBZ-                                       propylamino-(amido)N--[3-((S)-1'-phenylethyl)aminopropyl]-BLM                                                  PEP                                    48    3-{N--methyl-N--benzyl-N--[3-dibenzylamino)proplyamino}-                                                       dMTBZ-                                       propylamino-(amido)N--[octyl]-BLM                                                                              OCT                                    49    3-{N--methyl-N--benzyl-N--[3-(dibenzylamino)propylamino}-                                                      dMTBZ-                                       propylamino-(amido)N--{3-{N--methyl-N--benzyl-N--[3-dibenzyl-                                                  MTBZ                                         amino)propyl]amino}propyl}-BLM                                          50    3-{N--methyl-N--benzyl-N--[3-(dibenzylamino)propyl]amino}-                                                     dMTBZ-                                       propylamino-(amido)N--[3-[N--methyl-N--(3-n-butylaminopropyl)]-                                                BAPP                                         aminopropyl]-BLM                                                        51    3-{N--methyl-N--benzyl-N--[3-(dibenzylamino)propyl]amino}-                                                     dMTBZ-                                       propylamino-(amido)N--[benzyl]-BLM                                                                             BA                                     52    3-{N,N--dimethyl-N--[dibenzylamino)propyl]amino}-                                                              dMMDBZ-                                      propylamino-(amido)N--[3-((S)-1'-phenyl)aminopropyl]-BLM                                                       PEP                                    53    3-{N,N--dimethyl-N--[3-(dibenzylamino)propyl]amino}-                                                           dMMDBZ-                                      propylamino-(amido)N--[n-octyl]-BLM                                                                            OCT                                    54    3-{N,N--dimethyl-N--[3-dibenzylamino)propyl]amino}-                                                            dMMDBZ-                                      propylamino-(amido)N--[3-(di-n-butylamino)propyl]-BLM                                                          BPA                                    55    3-{N,N--diethyl-N--[3-(dibenzylamino)propyl]amino}-propyl-                                                     dEEDBZ-                                      amino-(amido)N--[3-((S)-1'-phenylethyl)aminopropyl]-BLM                                                        PEP                                    56    3-{N,N--diethyl-N--[3-(dibenzylamino)propyl]amino}propylamino-                                                 dEEDBZ-                                      (amido)N--[octyl]-BLM            OCT                                    57    3-{N,N--diethyl-N--[3-(dibenzylamino)propyl]amino}propylamino-                                                 dEEDBZ-                                      (amido)N--[3-(di-n-butylamino)propyl]-BLM                                                                      BPA                                    58    3-[N--methyl-N--[3-(cyclooctylmethylamino)propyl]amino}-                                                       dMCO-                                        propylamino-(amido)N--[3-(di-n-butyamino)propyl]-BLM                                                           BPA                                    59    3-{N--methyl-N--[3-(cyclooctylmethylamino)propyl]amino}propyl-                                                 dMCO-                                        amino-(amido)N--[octyl]-BLM      OCT                                    60    3-{N--methyl-N--[3-(cyclooctylmethyl)aminopropyl]amino}-                                                       dMCO-PEP                                     propylamino-(amido)N--[3-((S)-1'-phenylethylamino)propyl]-BLM           61    3-{N--methyl-N--[3-(p-cyanobenzylamino)propyl]amino}-                                                          dMCNBZ-                                      propylamino-(amido)N--[3-(di-n-butylamino)propyl]-BLM                                                          BPA                                    62    3-{N--methyl-N--[3-(p-cyanobenzylamino)propyl]amino}propylamino-                                               dMCNBZ-                                      (amido)N--[octyl]-BLM            OCT                                    63    3-{N--methyl-N--[3-(p-cyanobenzylamino)propyl]amino]propylamino-                                               dMCNBZ-                                      (amido)N--[(3-((S)-1'-phenylethylamino)propyl]-BLM                                                             PEP                                    64    3-{N--methyl-N--[3-bis(m,p-dibenzyloxybenzyl)aminopropyl]amino}-                                               dMDDBZOBZ-                                   propylamino-(amido)N--[3-((S)-1'-phenylethyl)aminopropyl]-BLM                                                  PEP                                    65    3-{N--methyl-N--[3-bis(m,p-dibenzyloxybenzyl)aminopropyl]amino}-                                               dMDDBZOBZ-                                   propylamino-(amido)N--[n-octyl]-BLM                                                                            OCT                                    __________________________________________________________________________

The present compounds represented by the genera-formula (I) are preparedas described below.

A compound represented by the following formula (VI) or reactivederivative of the carboxyl group thereof is allowed to condense with anamine (described later): ##STR16## wherein BM represents a moiety ofbleomycin skeleton, R₀ represents a hydroxyl group or a terminal aminoresidue of bleomycins, X₀ represntes a hydroxyl group or a group of theformula --NH--X, at least either one of R₀ and X₀ being the hydroxylgroup, and X represents (1) an alkyl of 1 to 18 carbon atoms, (2) anaminoalkyl of 1 to 12 carbon atoms, (3) a lower alkyl having assubstituent (a) 1 to 3 halogen atoms, (b) 1 or 2 phenyl groups, (c) anindolyl group, or (d) a 5- or 6-membered heterocyclic group containingan oxygen, sulfur or nitrogen atom (among the substituent groups, thephenyl or indolyl group may be further substituted by a halogen atom ora lower alkoxy group), (4) X₁ -lower alkyl [where X₁ is ##STR17## X₂ isa hydrogen atom, a lower alkyl or benzyl, X₃ is (a) a lower alkyl, (b) aphenyl(lower)alkyl, or (c) a moro- or di-(lower)alkylamino(lower)alkylwhich may be substituted by a phenyl or halophenyl group, X₄ is (a) alower alkyl or (b) a phenyl(lower)alkyl], (5) naphthyl, (6) thiazolyl,or (7) an N-phenyl(lower)alkylpiperazinyl. When X₀ in the formula (VI)is the hydroxyl group, an amine of the formula

    H.sub.2 N--X                                               (VII)

(wherein X is as defined above) is used in the condensation, while whenR₀ in the formula (VI) is the hydroxyl group and X₀ is --NH--X (where Xis as defined above), an amine of the formula

    H--R                                                       (VIII)

(where R is as defined above) is used. If necessary, the condensationproduct is removed of the copper to obtain an (amido)N-substitutedbleomycin of the general formula (I). Further, if necessary, a salt ofthe (amido)N-substituted bleomycin is prepared in a customary way.

The condensation of a compound of the formula (VI) or a reactivederivative of its carboxyl group with an amine of the formula (VII) or(VIII) is conducted according to the known procedure for forming anacidamide linkage, especially that used in peptide chemistry. A compoundof the formula (VI) is allowed to react with an amine of the formula(VII) or (VIII) in the presence of those activation reagents forcarboxyl group which are used in peptide chemistry. Alternatively, thecompound of formula (I) is obtained by the reaction of an amine of theformula (VII) or (VIII) with a reactive derivative of the carboxyl groupof a compound of formula (VI). The derivatives are, for example, aderivative obtained by reaction of an activation reagent for thecarboxyl group and the compound of formula (VI) or a 3-aminopropyl esterof the carboxyl group of the compound of formula (VI). As examples ofthe activation reagents, mention may be made of6-chloro-1-p-chlorobenzenesulfonyl-oxybenzotriazole,N-ethyl-5-phenylisooxazolium-3'-sulfonate,N-tert-butyl-5-methylisooxazolium perchlorate,N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (FEDQ), di-p-nitrophenylsulphite, tri-p-nitrophenyl phosphite, p-nitrophenyl trichloroacetate,dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, diphenylcarbodiimide,di-p-tolylcarbodiimide, diisopropylcarbodiimide, diphenylphosphorazidate(DPPA), and diethyl phosphorocyanidate (DEPC).

As examples of the reactive derivatives of carboxyl group of thecompounds of formula (VI), mention may be made of reactive derivativesobtained by the reaction of the above activation reagents with thecarboxyl group; reactive derivatives of carboxyl group obtained by thejoint use of the activation reagents and the condensation additives suchas, for example, p-nitrophenol, o,p-dinitrophenol, pentachlorophenol,2,4,5-trichlorophenol, pentafluorophenol, N-hydroxysuccinimide,1-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-dicarboximide;3-aminopropyl ester of the carboxyl group of formula (VI), andmonosubstituted 3-aminopropyl esters such as, for example,3-acetylaminopropyl ester, 3-succinylaminopropyl ester,3-benzoylaminopropyl ester, 3-benzylaminopropyl ester,3-p-toluenesulfonylaminopropyl ester, 3-(2,4-dinitrophenyl)aminopropylester, 3-(3,5-dimethyl-3-oxocyclohexen-1-yl)aminopropyl ester,3-(tert-butoxycarbonyl)aminopropyl ester, and3-(salicylidene)iminopropyl ester.

The condensation is carried out generally in a solvent. The solvent maybe any of those which will not affect the reaction, but it is preferredto use a polar solvent which dissolves the compound of formula (VI) usedas a starting material. Examples of such solvents are water,dimethylformamide, dimethylacetamide, acetonitrile, and mixturesthereof. The molar ratio of the amine of formula (VII) or (VIII) to thecompound of formula (VI) is generally about 0.5 to 20, preferably about1 to 10. Although the reaction temperature depends upon the type ofsolvent and other conditions, yet it is generally -15° to 45° C.,preferably -10° to 30° C. The reaction time is 1 to 70 hours. Theprocess according to this invention is described below in detail.

The copper-containing form of the compound of formula (VI) and, ifnecessary, the above-noted additive are dissolved in water,dimethylformaldehyde, acetonitrile, or a mixture thereof. To thesolution is added the above-noted activation reagent while stirring at-5° to 15° C. If necessary, the mixture is adjusted to a pH suitable foractivation with an inorganic acid or base such as hydrochloric acid orsodium hydroxide or with an organic base such as triethylamine orN-methylmorpholine. To the mixture is then added the amine component assuch or in a solution of adjusted pH. The resulting mixture is stirredand, if necessary, pH is adjusted in a similar manner to that describedabove. The stirring is contained for 1 to 70 hours to yield the intendedcompound of this invention.

Isolation of the derivative thus formed is performed in the followingmanner. As organic solvent such as acetone or ether is added to thereaction mixture to precipitate the intended product. The precipitate isdissolved in distilled water, then adjusted to pH 6, and, for thepurpose of desalting, is passed through a column packed with anadsorptive resin such as, for example, Amberlite®XAD-2 (Rohm and HaasCo.) in distilled water to effect adsorption of the intended product tothe resin. The column is washed with distilled water to remove the salt,and eluted with an acidified aqueous methanol such as a mixture of 1/50Nhydrochloric acid and methanol (1:4 v/v) to collect the fractions havingan absorption maximum at around a wave length of 290 mμ. The combinedfraction is neutralized with Dowex®44 (an OH-type anion exchange resinof Dow Chemical Co.), then concentrated under reduced pressure, andlyophilized to obtain a crude powder of the derivative. In some cases,the desalting step may be omitted and the solution of the precipitate indistilled water can be directly subjected to the second step oftreatment.

In the second step, the solution of crude powder in distilled water ispassed through a column packed with CM-Sephadex®C-25 (Na⁺ type;Pharmacia Fine Chemicals Co.), which has been equalibrated with a 1/20Macetic acid-sodium acetate buffer solution (pH 4.5), to effectadsorption of the intended product. The adsorbed phase is then eluted bythe linear concentration gradient technique which is carried out bycontinuously adding sodium chloride to the above buffer solution toincrease the sodium chloride concentration gradually to 1.0M. In thisstep, the unreacted reactants and the by-products tend to be eluted inearlier stage and can be separated by means of an ultraviolet absorptionmonitor. If the fraction of the intended product is found to becontaminated with impurities, the chromatography is repeated to removethe entire impurities.

Alternatively, in place of the above chromatography, another method ofchromatographic purification can be performed by employing an adsorptiveresin Amberlite®XAD-2 for example. The aqueous solution of the crudeproduct is passed through the column packed with the resin in a buffersolution such as, for example, 1% aqueous ammonium acetate solution toeffect adsorption of the intended product. The adsorbed phase is elutedby the linear concentration gradient technique in which methanol iscontinuously added to the buffer solution to increase gradually themethanol concentration. Since the unreacted reactants tend to be elutedin earlier stage and the main by-products in later stage, they can beseparated by use of an ultraviolet monitor. If the intended fractionsare found to be contaminated with impurities, they can be completelyremoved by repeating the chromatography.

The above two types of chromatography are performed each alone or incombination. The purified fraction containing the intended product isdesalted by use of the adsorptive resin such as, for example,Amberlite®XAD-2, and lyophilized to yield a blue amorphous powder of acopper-containing (amido)N-substituted bleomycin. The copper-free formis obtained by removing the copper from the copper-containing form by aknown method such as the method employing EDTA as disclosed in JapanesePatent Publication No. 31,875/77. An example of the copper-removingprocedure is described below.

The copper-containing product is dissolved in distilled water and theresulting solution is passes through a column packed withAmberlite®XAD-2 in distilled water to effect adsorption of thecopper-containing product. The resin is then washed with an aqueoussolution containing sodium chloride and 5% of disodium salt ofethylenediaminetetraacetic acid (briefly EDTA.2Na) to carry away thecopper ion by EDTA.2Na, leaving behind the copper-free(amino)N-substituted bleomycin adsorbed to the resin. The resin is thenwashed with a sodium chloride solution to remove EDTA.2Na, then withdistilled water, and finally eluted with an acidified aqueous methanolsuch as, for example, a 1/50N aqueous hydrochloric acid-methanol (1:4v/v) mixture to collect the fractions which show an absorption maximumat around 290 mμ. The combined fraction is adjusted to pH 6.0 withDowex®44 (OH type; Dow Chemical Co.), concentrated under reducedpressure, and lyophilized to yield a white amorphous powder of acopper-free (amido)N-substituted bleomycin hydrochloride. If sodiumsulfate and aqueous sulfuric acid are used in place of the sodiumchloride and aqueous hydrochloric acid, there is obtained a sulfate.Thus, any desired salt can be obtained by selecting the salt and acidused in the elution step. As examples of salts obtained in such amanner, there may be listed acetate, tartarate, citrate, maleate andlactate in addition to the hydrochloride and sulfate.

When the (amido)N-substituted bleomycin prepared as described above issubjected to hydrolysis with 6N aqueous hydrochloric acid at 105° C. for20 hours, there are detected, besides the amine R--H and X--NH₂ or adecomposition product of said amine in some cases, those decompositionproducts which are common to bleomycins, including L-threonine,β-amino-β-(4-amino-6-carboxy-5-methyl-pyrimidin-2-yl)propionic acid,4-amino-3-hydroxy-2-methyl-n-pentanoic acid, β-hydroxy-L-histidine,β-amino-L-alanine, and 2'-(2-aminoethyl)-2,4'-bithiazole-4-carboxylicacid. This fact supports that the (amido)N-substituted bleomycin has thechemical structure represented by the aforementioned formula (I).

In the general formula (VI) of compounds used as starting material, whenX₀ is a hydroxyl group and R₀ is a terminal amino residue of bleomycins,there is obtained a deamidobleomycin (copper-containing form) of theformula ##STR18## wherein BM and R are as defined previously. It isprepared by hydrolyzing copper-free bleomycins of the general formula(II) with an inactivating enzyme of rat or that similarly extracted frombovine or swine liver.

For instance, a bovine liver is homogenized in a phosphate buffersolution and centrifuged at 8,000 rpm. The supernatant is dialyzedagainst a phosphate buffer to obtain a crude enzyme solution. To thisenzyme solution is added a solution of bleomycins in a phosphate buffer.The mixture is allowed to react at 37° C. for 5 to 48 hours and thereaction mixture is removed of the protein by suitable means [theprotein can be removed, for example, by adding trichloroacetic acid(briefly TCA) to a concentration of 5% to precipitate the protein,separating the precipitate by centrifugation, and washing theprecipitate three times with a 5% TCA solution to collect the washings].The protein-free mixture is neutralized, mixed with copper acetate inexcess to convert the intended product into a copper-chelate. For thepurpose of desalting, the copper-chelate is passed through a columnpacked with an adsorptive resin, Dowex®HP 40, in distilled water toeffect adsorption of the intended product. The salts are washed off withdistilled water and the adsorbed phase is eluted with a 1/50Nhydrochloric acid-methanol (1:4 v/v) mixture to collect fractions whichshow an absorption maximum at around 290 mμ. The collected fraction isneutralized with an anion exchange resin, Dowex®44 (OH type, DowChemical Co.), concentrated under reduced pressure, and lyophilized. Theresulting powder is dissolved in distilled water and passed through acolumn packed with CM-Sephadex®C-25 (Na⁺ type; Pharmacia Fine ChemicalsCo.) which has been equilibrated with a 1/20M acetic acid-sodium acetatebuffer solution of pH 4.5 to effect adsorption. The adsorbed phase iseluted by the linear concentration gradient method in which sodiumchloride is continuously added to the above buffer solution to increasegradually the sodium concentration to 1.0M. The eluate fractions, bluein color, containing the intended product are collected, desalted by theDiaion®HP 40 desalting method as described above, and lyophilized toyield a blue amorphous powder of copper-containing deamidobleomycins.

When X₀ is --NH--X (X is as defined previously) and R₀ is OH in thegeneral formula (VI) of the compounds used as starting materials, theformula becomes that of an (amido)N-substituted bleomycinic acid;##STR19## wherein BM is as defined previously. An example of the methodof its synthesis is given below.

Using the above-mentioned inactivating enzyme, bleomycin B₂, a knowncompound, is converted into deamidobleomycin B₂, a deamidobleomycin ofthe formula (VI) in which R₀ is agmantine. This deamidobleomycin isallowed to react with an amine of the formula (VII) as described aboveto form an (amido)N-substituted bleomycin B₂ in which R₀ is agmantine,and hydrolyzing the resulting compound by use of a known fungal mycelium[for example, IFO 8502 deposited in Institute for Fermentation, Osaka]to yield the intended product.

Deamidobleomycinic acid represented by the formula ##STR20## is astarting material of the general formula (VI) in which both X₀ and R₀are hydroxyl groups. It is produced by hydrolyzing a known bleomycinicacid (see U.S. Pat. No. 3,886,133) with the aforementioned inactivatingenzyme. It is also obtained by hydrolyzing deamidobleomycin B₂ of thegeneral formula (VI-1), in which R is agmantine, by using theabove-mentioned fungus cell.

As examples of the amines of general formula (VII), mention may be madeof methylamine, ethylamine, n-propylamine, isopropylamine, n-butylamine,isobutylamine, tert-butylamine, n-penthylamine, 3-methylbutylamine,neopentylamine, n-heptylamine, 3-methylpentylamine, n-hexylamine,1,5-dimethylhexylamine, isohexylamine, n-octylamine, n-decylamine,laurylamine, myristylamine, cetylamine, stearylamine, 2-aminoethylamine,3-aminopropylamine, 4-aminobutylamine, 6-aminohexylamine,12-aminododecylamine, 4-amino-4-methyl-1-dimethylpentylamine,2,2,2-trifluoroethylamine, 2,2,2-trichloroethylamine,2,2-difluoroethylamine, 2,2-dichloroethylamine, benzylamine,diphenylmethylamine, 2-phenylethylamine, 2,2-diphenylethylamine,1-phenylethylamine, 1,2-diphenylethylamine, 3-phenylpropylamine,2-phenylisopropylamine, 1,3-diphenylpropylamine,3,3-diphenylpropylamine, 4-phenylbutylamine, 4,4-diphenylbutylamine,chlorobenzylamine, dichlorobenzylamine, bromobenzylamine,methoxybenzylamine, dimethoxybenzylamine, ethoxybenzylamine,methylenedioxybenzylamine, propoxybenzylamine, p-chlorophenylethylamine,p-methoxyphenylethylamine, p-benzyloxybenzylamine, furylmethylamine,2-furylethylamine, 2-thiazolylmethylamine, 2-pyrazolylmethylamine,2-imidazolylmethylamine, 4-imidazolylmethylamine, 2-thienylmethylamine,2-pyridylmethylamine, 3-pyridylmethylamine, 4-pyridylmethylamine,indolylmethylamine, indolylethylamine, 4-methoxyindolylmethylamine,2-pyrimidylmethylamine, 2-pyrimidylethylamine, 4-pyrimidylmethylamine,4-pyrimidylethylamine, 2-piperidylmethylamine, 3-piperidylmethylamine,4-piperidylmethylamine, 2-(2-piperidyl)ethylamine,2-(3-piperidyl)ethylamine, 2-(4-piperidyl)ethylamine,1-(2-piperidyl)ethylamine, 1-(3-piperidyl)ethylamine,1-(4-piperidyl)ethylamine, 2-(piperidino)ethylamine,2-piperazylmethylamine, 2-(2-piperazylethyl)amine,2-(piperidino)ethylamine, 3-piperidinopropylamine,2-(morpholino)ethylamine, 3-(morpholino)propylamine,2-morpholinylmethylamine, 3-morpholinylmethylamine,2-(morpholinyl)ethylamine, 3-(morpholinyl)propylamine,dimethylaminoethylamine, diethylaminoethylamine,dipropylaminoethylamine, propylaminoethylamine,dimethylaminopropylamine, diethylaminopropylamine,dipropylaminopropylamine, propylaminopropylamine,dibutylaminopropylamine, butylaminopropylamine, benzylaminopropylamine,2-phenylethylaminopropylamine, 1-phenylethylaminopropylamine,3-phenylpropylaminopropylamine, 4-phenylbutylaminopropylamine,methylaminoethylaminopropylamine, ethylaminopropylaminopropylamine,propylaminopropylaminopropylamine, butylaminopropylaminopropylamine,N-(butylaminopropyl)-N-methylaminopropylamine,dibutylaminopropylaminopropylamine, pentylaminopropylaminopropylamine,ethylaminobutylaminopropylamine, propylaminobutylaminopropylamine,ethylaminopropylaminobutylamine, butylaminopropylaminobutylamine,benzylaminoethylaminoethylamine, benzylaminoethylaminopropylamine,benzylaminopropylaminopropylamine, benzylaminobutylaminopropylamine,phenylethylaminopropylaminopropylamine,N-(phenylethylaminopropyl)-N-methylaminopropylamine,N-(chlorobenzylaminopropyl)-N-methylaminopropylamine,N-(bromobenzylaminopropyl)-N-methylaminopropylamine,N-(chlorophenylethylaminopropyl)-N-methylaminopropylamine,dibenzylaminopropyl-N,N-dimethylaminopropylamine,dibenzylaminopropyl-N,N-diethylaminopropylamine,dibenzylaminopropyl-N-methyl-N-benzylaminopropylamine, α-naphthylamine,β-naphthylamine, 2-thiazolylamine, 3-thiazolylamine, 4-thiazolylamine,N-benzyl-4-piperidylamine, N-phenylethyl-4-piperidylamine,N-benzyl-3-piperidylamine, and N-benzyl-2-piperidylamine.

Desirable amines of formula (VIII) are those aliphatic primary amineswhich have a basic group in addition to the amino group participating inthe reaction. As examples of such amines, mention may be made of2-aminoethylamine, 3-aminopropylamine, 2-dimethylaminoethylamine,2-diethylaminoethylamine, 3-dimethylaminopropylamine,3-diethylaminopropylamine, 3-(3-butylaminopropylamino)propylamine,3-(2-hydroxypropylamino)propylamine, 3-piperidinopropylamine,3-(1-phenylethylamino)propylamine, 2-aminopropylamine,3-methylaminopropylamine, 3-butylaminopropylamine,3-(6-aminohexylamino)propylamine, 3-trimethylaminopropylamine,3-(3-dimethylaminopropylamino)propylamine,3-(3-aminopropylamino)propylamine,3-[N-methyl-N-(3-aminopropyl)amino]propylamine,3-pyrrolidinylpropylamine, 3-piperidinylpropylamine,3-morpholinopropylamine, 3-piperazinylpropylamine,3-[4-(3-aminopropyl)piperazinyl]propylamine-3-(3-pyrrolidinylpropylamino)propylamine,3-(3-piperidinylpropylamino)propylamine,3-(3-morpholinylpropylamino)propylamine,3-(3-hydroxypropylamino)propylamine,3-(3-methoxypropylamino)propylamine, 3-benzylaminopropylamine,m-aminomethylbenzylamine, p-aminomethylbenzylamine,2-cyclopentylaminoethylamine, 3-cyclohexylaminopropylamine,4-cyclohexylaminobutylamine, cycloheptylaminopropylamine,3-cyclooctylaminopropylamine,3-[N-methyl-3-N-(cyclooctylmethylaminopropyl)amino]propylamine,3-cyclodecanylmethylaminopropylamine,3-{N-methyl-N-[3-(2-p-chlorophenylethylamino)propyl]amino}propylamine,3-{N-methyl-N-[3-(m,p-dibenzyloxybenzyl)aminopropyl]amino}propylamine,3-{N-methyl-N-[3-(p-cyanobenzylamino)propyl]amino}propylamine,3-{N-methyl-N-[3-(cycloundecanylmethylamino)propyl]amino}propylamine,3-{N-methyl-N-[bis(m,p-dibenzyloxybenzyl)aminopropyl]amino}propylamine,3-{N,N-dimethyl-N-[3-(dibenzylamino)propyl]amino}propylamine,3-{N,N-diethyl-N-[3-(dibenzylamino)propyl]amino}propylamine,3-{N,N-dimethyl-N-[3-(N,N-dimethyl-N-(3-dibenzylaminopropyl)amino)propyl]amino}propylamine,3-{N,N-dimethyl-N-[3-(N,N-dimethyl-N-(3-cyclooctylmethylaminopropyl)amino)propyl]amino}propylamine,3-[4-(3-dibenzylaminopropylamino)piperidyl]propylamine, and3-[4-(3-cyclooctylmethylaminopropyl)piperidyl]propylamine.

Among the amines of the general formula (VIII), those represented by thefollowing general formula are novel compounds first synthesized by thepresent inventors:

    NH.sub.2 --(CH.sub.2).sub.3 --A'--(CH.sub.2).sub.3 --B'    (IX)

wherein A' represents ##STR21## (where R₁₀ ' and R₁₁ ' are each a loweralkyl or benzyl and B' represents di[phenyl(lower)alkyl]amino,cyanophenyl(lower)alkylamino, C₅₋₁₃ -cycloalkyl-substituted loweralkylamino, or bis[dibenzyloxyphenyl(lower)alkyl]amino. These compoundsare synthesized by the hydrolysis of the compound represented by thegeneral formula

    Y--(CH.sub.2).sub.3 --A'--(CH.sub.2).sub.3 --B'            (X)

(wherein A' and B' are as defined above and Y is a protected aminogroup) to remove the protective group. Although the hydrolysis takesplace in the presence of either an acid or a base, yet an acid such ashydrochloric acid is generally preferred.

Of the starting materials of the general formula (X), those representedby the formula ##STR22## [wherein R₁₀ ', R₁₁ ' and Y are as definedabove and R₁₂ " and R₁₃ " are each a phenyl(lower)alkyl] are prepared inthe following manner.

A 3-aminopropyl-N,N-dialkylamine of the general formula ##STR23##(wherein R₁₀ ' and R₁₁ ' are as defined above) is subjected to reductivecondensation with benzaldehyde or a phenyl(lower)aldehyde. The reactionmixture is made alkaline and extracted with an organic solvent to obtainan N',N'-dibenzylaminopropyl-N,N-dialkylamine of the general formula##STR24## (wherein R₁₀ ', R₁₁ ', R₁₂ " and R₁₃ " are as defined above).The resulting amine is allowed to react with a3-halopropyl-N-(protected)amine to form a quaternary salt of the formula##STR25## wherein Y, R₁₀ ', R₁₁ ', R₁₂ " and R₁₃ " are as defined above.The quaternary salt is hydrolyzed in the presence of an acid, forexample, 6N hydrochloric acid, at a temperature of from room temperatureto 200° C., e.g. at 110° C. for 8 hours. After the removal ofby-products such as an acid,the reaction mixture is concentrated toyield a hydrochloride of a compound of the formula (XI).

An amine of the general formula ##STR26## wherein Y is as defined aboveand B" is a di[phenyl(lower)alkyl]amino, p-cyanophenyl(lower)alkylamino,C₅₋₁₁ -cycloalkyl-substituted lower alkylamino, orbis-[dibenzyloxyphenyl(lower)alkyl]amino, is prepared by acylating abis(3-aminopropyl) (lower)alkylamine with one equivalent or less of anacylating agent such as, for example, benzoyl chloride, acetyl chloride,acetic anhdride, carbobenzoxy chloride,S-tert-butoxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine, orN-carbethoxyphthalimide to form a monoacylated derivative, subjectingthe resulting derivative to reductive condensation with one equivalentof an aldehyde when B" is a monosubstituted amino group or with 2 ormore, preferably 3 to 6, equivalents of an aldehyde when B" is adisubstituted amino group, and hydrolyzing the acyl group withhydrochloric acid.

The physicochemical properties of the typical (amido)N-substitutedbleomycin derivatives of this invention are as shown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________                     UV absorbtion                                                                          TLC*.sup.1 of Cu--                                                   maximum of                                                                             containing                                                                            Electrophoresis*.sup.2                      Compound         Cu--free form,                                                                         form    of Cu--containing form,                     No.   Abbreviation                                                                             mμ (E 1%/1 cm)                                                                      Rf      Rm (Rm of alanine = 1.0)                    __________________________________________________________________________    1     dH-MMA      291 (111)                                                                             0.62*   0.92                                        2     dH-MEA      291 (107)                                                                             0.75    0.83                                        3     dH-IPA      291 (105)                                                                             0.56*   0.85                                        4     dH-BA      291 (98) 0.70    0.75                                        5     dH-PEP      291 (102)                                                                             0.68    0.96                                        6     dH-EDA      291 (100)                                                                             0.45    1.04                                        7     dB2-IPA    291 (89) 0.53*   0.93                                        8     dB2-DHA    291 (94) 0.76    0.87                                        9     dB2-TFEA   291 (93) 0.50*   0.85                                        10    dB2-OCT    291 (85) 0.72    0.77                                        11    dB2-PEP    291 (79) 0.87    1.03                                        12    dB2-BPA    292 (79) 0.87    1.02                                        13    dB2-LAA    291 (82) 0.43    0.70                                        14    dB2-BA     291 (78) 0.39*   0.78                                        15    dB2-ADPM   291 (89) 0.82    0.76                                        16    dB2-DPE    291 (81) 0.77    0.76                                        17    dB2-NA     291 (85) 0.84    0.83                                        18    dB2-FFA     292 (115)                                                                             0.48*   0.82                                        19    dB2-AMPY   292 (84) 0.47*   1.02                                        20    dB2-ATZ     292 (137)                                                                             0.86    0.78                                        21    dPEP-IPA   291 (94) 0.84    0.87                                        22    dPEP-PEP   291 (89) 0.77    0.98                                        23    dPEP-BA    291 (91) 0.78    0.87                                        24    dPEP-CBA   291 (81) 0.74    0.79                                        25    dPEP-STE   291 (68) 0.06    0.18                                        26    dPEP-LAA   291 (83) 0.33    0.75                                        27    dPEP-OCT   291 (83) 0.61    0.84                                        28    dPEP-MTA    285 (100)                                                                             0.75    0.81                                        29    dPEP-DAD   291 (94) 0.78    1.02                                        30    dPEP-DPE   291 (88) 0.65    0.87                                        31    dPEP-ADPM  291 (85) 0.69    0.87                                        32    dPEP-DHA   291 (91) 0.63    0.84                                        33    dPEP-APZ   291 (85) 0.78    1.26                                        34    dPEP-BPA   291 (82) 0.73    1.00                                        35    dPEP-ABP   291 (78) 0.72    1.10                                        36    dPEP-ADP   291 (75) 0.80    1.06                                        37    dBAPP-IPA  291 (91) 0.85    1.14                                        38    dBAPP-BAPP 291 (83) 0.26    1.44                                        39    dMCLBZ-PEP 289 (90) 0.53    1.16                                        40    dMCLBZ-OCT 290 (88) 0.50    1.08                                        41    dMCLBZ-DPE 290 (78) 0.55    1.01                                        42    dMCLBZ-DHA 290 (80) 0.53    0.90                                        43    dMCLPE-PEP 290 (79) 0.53    1.04                                        44    dMCLPE-OCT 290 (85) 0.52    0.96                                        45    dMCLPE-MCLPE                                                                             290 (79) 0.25    1.20                                        46    dMCLPE-BPA 290 (83) 0.60    1.11                                        47    dMTBZ-PEP  290 (81) 0.38    1.06                                        48    dMTBZ-OCT  290 (73) 0.24    0.92                                        49    dMTBZ-MTBA 290 (69) 0.15    1.07                                        50    dMTBZ-BAPP 290 (71) 0.47    0.79                                        51    dMTBZ-BA   290 (81) 0.48    0.79                                        52    dMMDBZ-PEP 291 (75) 0.50    1.15                                        53    dMMDBZ-OCT 291 (76) 0.35    1.01                                        54    dMMDBZ-BPA 291 (79) 0.49    1.13                                        55    dEEDBZ-PEP 291 (76) 0.48    1.12                                        56    dEEDBZ-OCT 291 (78) 0.33    1.00                                        57    dEEDBZ-BPA 291 (73) 0.48    1.14                                        58    dMCO-BPA   291 (87) 0.54    1.08                                        59    dMCO-OCT   291 (83) 0.42    0.96                                        60    dMCO-PEP   291 (89) 0.53    1.06                                        61    dMCNBZ-BPA 290 (81) 0.70    1.10                                        62    dMCNBZ-OCT 292 (94) 0.59    0.95                                        63    dMCNBZ-PEP 291 (93) 0.69    1.10                                        64    dMDDBZOBZ-PEP                                                                            285 (74) 0.09    0.93                                        65    dMDDBZOBZ-OCT                                                                            285 (76) 0.04    0.64                                        __________________________________________________________________________     Note:                                                                         *.sup.1 "Silica Gel 60F 254 Silanised ®" (Merck Co.); methanol6%          ammonium acetate (60:40 v/v) except for Rf values with an asterisk which      were measured in another mixture (65:34 v/v).                                 *.sup.2 Avicel SF ® (FMC Co.); formic acidacetic acidwater (27:75:900     v/v), 800 V, 15 minutes.                                                 

The biological properties, as determined on the typical compounds ofthis invention, are described below.

1. RESISTANCE TEST AGAINST INACTIVATING ENZYME

(1) Extraction of inactivating enzyme.

The liver of Donryu strain male rats was mixed with two times the weightof a 1/15M phosphate buffer solution of pH 7 and ground to prepare atissue emulsion. The resulting emulsion was centrifuged at 105,000 g(gravity constant) for 60 minutes. The supernatant was dialyzed and theresulting large molecule fraction was used as an inactivating-enzymeextract.

(2) Determination of inactivation reaction.

To 1 ml of the above extract, was added 1 ml of a substrate solutoincontaining 800 mcg of a bleomycin derivative. The mixture was allowed toreact at 37° C. for 40 minutes. A portion (0.3 ml) of the reactionmixture was removed of protein and tested for the residual activityagainst Mycobacterium smegmatis ATCC 607. Under the same conditions,bleomycin B₂, used as control, showed 50% decrease in activity. The testresults were as shown in Table 3. As is apparent from the test results,as compared with bleomycins, the (amido)N-substituted bleomycinderivatives of this invention are generally less affected by thebleomycin-inactivating enzyme.

2. ANTIMICROBIAL ACTIVITIES AGAINST MYCOBACTERIUM SMEGMATIS ATCC 607 ANDBACILLUS SUBTILIS

The antimicrobial activity was tested against the captioned testorganisms by the method of agar plate-cylinder. The activity wasrecorded on the assumption that the activity of standard sample ofbleomycin A₂ (copper-free form) is 1,000 mcg poteney/mg. The testresults were as shown in Table 3.

3. GROWTH INHIBITORY ACTIVITY AGAINST CULTURED HELA S₃ CELLS

HeLa S₃ cells were inoculated into a medium (MEM with 10% bovine serum)placed in a plastic petri dish. Two days thereafter, a bleomycin wasadded to the dish. After 3 days of breeding, the number of cells werecounted. The percentage growth inhibition was calculated using thefollowing equation:

Percentage inhibition (%)=100×(B-A)/(B-C) where A is the final number ofcells after 3 days from the addition of test sample, B is the finalnumber of cells in the control without addition of the test smaple, andC is the number of cells at the time of addition of the test sample.ID₅₀ (concentration of 50% inhibition) was estimated from the graphprepared by plotting the concentration of test sample against thepercentage inhibition. The results obtained were as shown in Table 3.

                                      TABLE 3                                     __________________________________________________________________________                     Antimicrobial potency                                                                      50% Growth in-                                                   of Cu--free compound                                                                       hibitory con-                                                    (mcg potency/mg)                                                                           centration of                                                                          Resistance of                                           Against      Cu--free compd.                                                                        Cu--free com-                                           Mycobacterium                                                                         Against                                                                            against cultured                                                                       pound against                          Compound         smegmatis                                                                             Bacillus                                                                           HeLaS.sub.3 cells                                                                      inactivating                           No.   Abbreviation                                                                             ATCC 607                                                                              subtilis                                                                           (ID.sub.50) mcg/ml                                                                     enzyme*.sup.1                          __________________________________________________________________________    1     dH-MMA     1447    654  1.2      ++++                                   2     dH-MEA     1895    767  0.98     +++                                    3     dH-IPA     2778    593  0.95     +++++                                  4     dH-BA      6595    579  1.10     +                                      5     dH-PEP     9846    3772 1.0      ++++                                   6     dH-EDA     2396    4453 1.0      ++++                                   7     dB2-IPA    6425    1950 0.56     ++++                                   8     dB2-DHA    24880   950  0.62     +++                                    9     dB2-TFEA   3990    730  1.03     ++                                     10    dB2-OCT    33700   1500 0.34     +                                      11    dB2-PEP    15729   7036 0.71     +++++                                  12    dB2-BPA    16438   4384 0.98     +++++                                  13    dB2-LAA    7650    660  0.39     ++++                                   14    dB2-BA     28140   1650 0.73     +                                      15    dB2-ADPM   42320   980  0.39     +++++                                  16    dB2-DPE    24480   520  0.67     ++++                                   17    dB2-NA     33650   1295 1.1      ±                                   18    dB2-FFA    19150   1285 0.58     ±                                   19    dB2-AMPY   5160    1010 0.83     ++                                     20    dB2-ATZ    4870    900  0.73     ++                                     21    dPEP-IPA   11998   962  0.17     ++++                                   22    dPEP-PEP   28030   2800 0.80     +++++                                  23    dPEP-BA    38348   1380 0.26     +                                      24    dPEP-CBA   56328   1184 0.28     +                                      25    dPEP-STE   210     0    4        *                                      26    dPEP-LAA   3920    384  0.40     ++++                                   27    dPEP-OCT   45100   886  0.36     ++                                     28    dPEP-MTA   18650   586  0.60     ++++                                   29    dPEP-DAD   14200   5400 0.43     ++++                                   30    dPEP-DPE   33802   190  1.02     ++++                                   31    dPEP-ADPM  54645   430  0.56     +++++                                  32    dPEP-DHA   39956   298  0.83     ++                                     33    dPEP-APZ   6359    5009 1.50     ++++                                   34    dPEP-BPA   26023   2843 0.95     ++++                                   35    dPEP-ABP   18537   2159 0.90     ++++                                   36    dPEP-ADP   6061    3694 2.70     + +++                                  37    dBAPP-IPA  13210   20048                                                                              0.90     ++++                                   38    dBAPP-BAPP 5100    6600 0.79     +++                                    39    dMCLBZ-PEP 14880   2590 0.53     +++++                                  40    dMCLBZ-OCT 38200   1808 0.43     +++                                    41    dMCLBZ-DPE 23342   521  0.86     +++++                                  42    dMCLBZ-DHA 31834   933  0.49     ++++                                   43    dMCLPE-PEP 15593   4267 0.37     +++++                                  44    dMCLPE-OCT 38750   1898 0.23     ++                                     45    dMCLPE-MCLPE                                                                             650     270  0.30     +++++                                  46    dMCLPE-BPA 31690   3690 0.40     +++++                                  47    dMTBZ-PEP  5900    180  2.15     +++++                                  48    dMTBZ-OCT  2580    130  1.75     *                                      49    dMTBZ-MTBZ 230     1    11.0     *                                      50    dMTBZ-BAPP 1417    376  2.2      ++++                                   51    dMTBZ-BA   24461   410  0.77     +++                                    52    dMMDBZ-PEP 21371   2372 0.55     +++++                                  53    dMMDBZ-OCT 40703   595  0.23     *                                      54    dMMDBZ-BPA 32633   2663 0.80     ++++                                   55    dEEDBZ-PEP 20840   2150 1.00     +++++                                  56    dEEDBZ-OCT 41380   520  0.60     *                                      57    dEEDBZ-BPA 25924   1831 1.5      +++ +                                  58    dMCO-BPA   62418   5518 0.40     ++++                                   59    dMCO-OCT   65733   5473 0.23     +++                                    60    dMCO-PEP   54345   7273 0.33     +++++                                  61    dMCNBZ-BPA 14730   4755 1.10     ++++                                   62    dMCNBZ-OCT 49270   381  0.28     +++                                    63    dMCNBZ-PEP 21877   11056                                                                              0.60     ++++                                   64    dMDDBZOBZ-PEP                                                                            115     23   0.058    *                                      65    dMDDBZOBZ-OCT                                                                            153     13   0.30     *                                      __________________________________________________________________________     Note:                                                                         *.sup.1 The resistance was rated in terms of the degree of inactivation       under the conditions under which the degree of inactivation of bleomycin      B.sub.2 is 50%.                                                          

    Rating                                                                              Degree of inactivation                                                  ±  45% or more                                                             +     35 to 44%                                                               ++    25 to 34%                                                               +++   15 to 24%                                                               ++++  5 to 14%                                                                +++++ less than 5%                                                            *     Undeterminable because                                                        of the adsorption to                                                          the enzyme protein.                                                 

4. PULMONARY TOXICITY (PULMONARY FIBROSIS) IN MICE

ICR strain mice (male, 15 weeks old) in groups of 9 members were used.Each test preparation was administered by intraperitoneal injection oncea day for 10 consecutive days at a dose rate of 5 mg/kg. Aftercompletion of the administration, the mice were bred for 5 weeks underobservation, then slughtered and autopsied to examine the incidence andgrade of the pulmonary fibrosis. The evaluation was made by comparingthe number of administered mice suffering from pulmonary fibrosis andthe grade of the disease. The results were as shown in Table 4. Thegrade was numerically rated as follows:

Number of points

0: No fibrosis.

1: Accumulation of exudate in alveolus and fibrosis-like change inalveolar septum.

2: Fibrosis in several areas.

4: Scattered fibrosis.

6: Fibrosis in more than two-thirds of the total area.

The "ratio" in Table 4 was calculated by comparison with "BleomycinComplex."

                                      TABLE 4                                     __________________________________________________________________________                             Grade                                                                 Incidence                                                                             Total score of                                                        Number of mice                                                                        pulmonary fibrosis/                                                   with pulmonary                                                                        total number of                                      Compound         fibrosis                                                                              samples                                              No.   Abbreviation                                                                             (%)     (%)       Ratio                                      __________________________________________________________________________    21    dPEP-IPA   3/8 (38)                                                                              5/24 (21) 0.27                                       22    dPEP-PEP   1/8 (13)                                                                              1/21 (4)  0.05                                       33    dPEP-APZ   5/9 (56)                                                                              11/27                                                                              (41) 0.44                                       34    dPEP-BPA   0/8 (0) 0/24 (0)  0                                          37    dBAPP-IPA  3/9 (33)                                                                              13/27                                                                              (48) 0.46                                       38    dBAPP-BAPP 5/7 (71)                                                                              7/21 (33) 0.32                                       39    dMCLBZ-PEP 2/9 (20)                                                                              2/27 (7)  0.07                                       43    dMCLPE-PEP 0/1 (0) 0/3  (0)  0                                          45    dMCLPE-MCLPE                                                                             0/9 (0) 0/27 (0)  0                                          47    dMTBZ-PEP  0/8 (0) 0/24 (0)  0                                          48    dMTBZ-OCT  0/8 (0) 0/24 (0)  0                                          49    dMTBZ-MTBZ 0/3 (0) 0/9  (0)  0                                          51    dMTBZ-BA   0/8 (0) 0/24 (0)  0                                          52    dMMDBZ-PEP 0/9 (0) 0/27 (0)  0                                          53    dMMDBZ-OCT 0/9 (0) 0/27 (0)  0                                          54    dMMDBZ-BPA 0/9 (0) 0/27 (0)  0                                          55    dEEDBZ-PEP 0/9 (0) 0/27 (0)  0                                          56    dEEDBZ-OCT 0/9 (0) 0/27 (0)  0                                          57    dEEDBZ-BPA 0/9 (0) 0/27 (0)  0                                          58    dMCO-BPA   0/9 (0) 0/27 (0)  0                                          59    dMCO-OCT   0/9 (0) 0/27 (0)  0                                          60    dMCO-PEP   0/9 (0) 0/27 (0)  0                                          62    dMCNBZ-OCT 0/9 (0) 0/27 (0)  0                                          64    dMDDBZOBZ-PEP                                                                            0/9 (0) 0/27 (0)  0                                          65    dMDDBZOBZ-OCT                                                                            0/9 (0) 0/27 (0)  0                                          __________________________________________________________________________

5. LD₅₀ IN MICE, AS DETERMINED BY ADMINISTRATION FOR 10 CONSECUTIVE DAYS

CDF₁ /SLC strain male mice (6 weeks old, 7 members per group) weresubcutaneously administered with various doses of bleomycin derivativesonce a day for 10 consecutive days. From the mortality during theadministration period, LD₅₀ (daily dose) was determined by the method ofBehrens-Karber. The results were as shown in Table 5.

                  TABLE 5                                                         ______________________________________                                                               LD.sub.50 during administra-                           Compound               tion period of 10 con-                                 No.     Abbreviation   secutive days. (mg/kg/day)                             ______________________________________                                        52      dMMDBZ-PEP     13.9                                                   53      dMMDBZ-OCT     15.0                                                   64      dMDDBZOBZ-PEP  >81.4                                                  65      dMDDBZOBZ-OCT  >75.7                                                  ______________________________________                                    

As is apparent from the foregoing description, the compounds of thepresent invention are resistant to a beomycin-inactivating enzyme, havea high growth-inhibitory activity against cultured HeLa S₃ cells as wellas a distingished antimicrobial activity, and are very low in pulmonarytoxicity, positively suggesting their usefulness in clinical fields.

When used as a drug, the compounds of this invention are mixed with anexcipient in a customary way and prepared in the form of injections,tablets, ointments, suppositories, and so on. Suitable excipientsinclude water, sugars such as mannitol, and other materials usually usedin medical preparations.

Although the dose varies depending on the administration form, a singledose of 2-10 mg per person is administered one to four times a week, thecumulative dose being 2-200 mg/person/week.

The invention is illustrated below in detail with reference to Examples,but the invention is not limited thereto.

EXAMPLE 1 Step A

Fresh bovine liver, 200 g in weight, was homogenized in 400 ml of a0.05M phosphate buffer solution of pH 7.2 and centrifuged at 8,000 rpmfor 30 minutes. The supernatant was dialyzed against 0.05M phosphatebuffer to prepare a crude enzyme solution. To 10 g of bleomycin B₂, wasadded 400 ml of the crude enzyme solution. The mixture was allowed toreact at 37° C. for 24 hours. To the reaction mixture, was added 40 mlof a 55% solution of trichloroacetic acid (briefly TCA) to precipitatethe protein. The precipitate was separated by centrifuging and washedthree times with 5% TCA solution. The supernatant and the washings werecombined, neutralized with a 4M sodium hydroxide solution, and admixedwith 3.2 g (2.4 equivalents to bleomycin) of copper acetate to form acopper-chelate of the intended product. For the purpose of desalting,the copper-chelate solution was passed through a column, 1 liter involume, packed with an adsorptive resin Diaion®HP 40 (MitsubishiChemical Co.) in distilled water, to effect the adsorption of intendedproduct. After washing off the salts with 1.5 liters of distilled water,the adsorbed phase was eluted with a 1/50N aqueous hydrochloricacid-methanol (1:4 v/v) mixture to collect the fractions which show anabsorption maximum at around a wave length of 290 mμ. The combinedfraction was neutralized with Dowex®44 (OH type, Dow chemical Co.) andconcentrated under reduced pressure. The concentrate was passed througha column, 1 liter in volume, packed with CM-Sephadex®C-25 (Na⁺ type,Pharmacia Fine Chemicals Co.), which had been equilibrated with a 1/20Macetic acid-sodium acetate buffer solution of pH 4.5, to effectadsorption. The adsorbed phase was eluted by the linear concentrationgradient method in which sodium chloride was continuously added to theabove-noted buffer solution to increase gradually the sodiumconcentration to 1.0M. The blue fractions eluted at a sodiumconcentration of about 0.3M were collected, then desalted by usingDiaion®HP 40 as described above, and lyophilized to yield 8.5 g (83%yield) of a blue amorphous powder of copper-containing deamidobleomycinB₂.

The blue powder showed absorption maxima (E 1%/1 cm) at 242 mμ (138) and291 mμ (115), as determined in distilled water. The infrared absorptionmaxima (in wave number, cm⁻¹), as measured in KBr-tablet, were 3425,2975, 2940, 1720, 1640, 1575, 1460, 1420, 1400, 1375, 1280, 1260, 1240,1190, 1140, 1100, 1060, 1020, 760. Other physicochemical properties wereas shown in Table 6.

In a similar manner to that described above, by using belomycin A₂ '-C,3-((S)-1'-phenylethyl)aminopropylaminobleomycin, and3-(3-n-butylaminopropylamino)propylaminobleomycin, there were obtaineddeamidobleomycin A₂ '-C,3-((S)-1'-phenylethylamino)propylamino-deamidobleomycin, and3-(3-n-butylaminopropylamino)propylaminodeamidobleomycin, respectively.The physicochemical properties of these compounds were as shown in Table6.

                                      TABLE 6                                     __________________________________________________________________________                         UV absorption                                                                          TLC*.sup.1                                                                          Electrophoresis*.sup.2                                         maximum of Cu--                                                                        of Cu--                                                                             of Cu--containing                         Synthesized          containing form                                                                        containing                                                                          form, Rm (Rm of                           deamidobleomycin     mμ (E 1%/1cm)                                                                       form, Rf                                                                            alanine = 1.0)                            __________________________________________________________________________    Deamidobleomycin B.sub.2                                                                           242 (138)                                                                              0.95  0.72                                                           292 (115)                                                                              0.64*                                           Deamidobleomycin A.sub.2 '-C                                                                       242 (132)                                                                              0.75  0.62                                                           292 (110)                                                                              0.46*                                           3-((S)--1'-phenylethylamino)propyl-                                                                242 (147)                                                                              0.87  0.67                                      deamidobleomycin     292 (124)                                                                              0.40*                                           3-(3-n-butylaminopropylamino)propylamino-                                                          242 (142)                                                                              0.83  1.01                                      deamidobleomycin     292 (118)                                                                              0.55*                                           __________________________________________________________________________     Note:                                                                         *.sup.1 "Silica Gel 60F 254 Silanised ®" (Merck Co.); measured in         methanol6% ammonium acetate solution mixture (60:40 v/v) except for Rf        values with an asterisk which were measured in another mixture (65:34 v/v     *.sup.2 Avicel SF ® (FMC Co.); formic acidacetic acidwater                (27:75:900v/v); 800 V, 15 minutes.                                       

Step B

To a solution of 1 g of copper-containing deamidobleomycin B₂ obtainedin step A and 1.77 g of 1-hydroxybenzotriazole (briefly HOBT) in 10 mlof dimethylformamide, while being cooled at 0° C. and stirred, was added1.35 g (10 equivalents to bleomycin) of dicyclohexylcarbodiimide(briefly DCC). Five minutes thereafter, to the mixture were added 840 mg(5 equivalents to bleomycin) of 3-((S)-1'-phenylethylamino)propylaminehydrochloride and 0.72 ml of N-methylamorpholine. The resulting mixturewas allowed to react with stirring at room temperature for 16 hours. Tothe reaction mixture was added 10 times by volume of acetone toprecipitate the intended product. After thorough washing with acetone,the precipitate was dissolved in distilled water and passed through acolumn, 100 ml in volume, packed with CM-Sephadex®C-25 (Na⁺ type,Pharmacia Fine Chemicals Co.), which had been equilibrated with a 1/20Macetic acid-sodium acetate buffer solution of pH 4.5, to effectadsorption. The adsorbed phase was eluted by the linear concentrationgradient method in which sodium chloride was continuously added to theabove-noted buffer solution to increase gradually the sodiumconcentration to 1.0M. The blue fractions eluted at a sodiumconcentration of around 0.6M were collected. The combined fraction wasdesalted by using 100 ml of Diaion®HP-40 as described above, andlyophilized to yield 880 mg (76% yield) of a blue amorphous powder ofcopper-containing amido(N)-[3-((S)-1'-phenylethylamino)propyl]-bleomycinB₂. It showed absorption maxima (E 1%/1 cm) at 243 mμ (125) and 292 mμ(96), as measured in distilled water. The IR absorption (in wave number,cm⁻¹), as measured in KBr-table, were 3425, 2975, 2930, 1720, 1640,1580, 1575, 1550, 1455, 1430, 1400, 1370, 1290, 1240, 1190, 1130, 1095,1060, 1005, 980, 875, 760. Other physicochemical properties were asshown in Table 2.

In a similar manner to that described above, copper-containing forms of(amido)N-substituted bleomycin B₂ Nos. 7 to 20 shown in Table 2 weresynthesized by the reaction of copper-containing deamidobleomycin B₂with amines corresponding to the intended products.

Similarly, compound Nos. 1, 6, 21-38, 64 and 65 shown in Table 2 weresynthesized by the reaction of deamidobleomycin A₂ '-C,3-((S)-1'-phenylethyl)aminopropylamino-deamindobleomycin, and3-(3-n-butylaminopropylamino)propylamino-deamidobleomycin (eachdeamidobleomycin in copper-containing form) with amines corresponding tothe intended products.

Step C

Into 20 ml of distilled water, was dissolved 880 mg of thecopper-containing compound obtained in step B. For the purpose ofdesalting, the solution was passed through a column, 100 ml in volume,packed with Amberlite®XAD-2 in distilled water, to effect adsorption ofthe copper-containing compound. The resin was washed successively with300 ml of an aqueous solution containing sodium chloride and 5% ofEDTA.2Na, 100 ml of 2% aqueous sodium chloride solution, and 250 ml ofdistilled water. The resin was then eluted with a 1/50N hydrochloricacid-methanol (1:4 v/v) mixture to collect the fractions which show anabsorption maximum at a wave length of around 290 mμ. The combinedfraction was adjusted to pH 6.0 with Dowex 44 (OH type, Dow ChemicalCo.), then concentrated under reduced pressure, and lyophilized to yield790 mg (93% yield) of a white amorphous powder of copper-freee(amido)N-[3-((S)-1'-phenylethylamino)propyl]-bleomycin B₂ hydrochloride.UV absorption maximum and (E 1%/1 cm) were 291 mμ and (79),respectively. IR absorption maxima (in wave number, cm⁻¹), as measuredin KBr-tablet were 3425, 2950, 1720, 1640, 1555, 1450, 1400, 1360, 1320,1260, 1190, 1060, 880, 910, 805, 770, and 700. Other physicochemicalproperties were as shown in Table 2.

EXAMPLE 2 Step A

Fungal mycelium, 400 g in weight, obtained by cultivating Fusariumroseum IFO 7189 deposited in the Institute for Fermentation, Osaka, washomogenized in 4 limiters of a 1/20M phosphate buffer solution of pH7.5. To the homogenized mixture, was added a solution of 10 g ofcopper-containing (amido)N-[3-((S)-1'-phenylethylamino)propyl]-bleomycinB₂ in 1 liter of the same phosphate buffer solution as used above. Themixture was allowed to react at 37° C. for 20 hours. The reactionmixture was mixed with a filter aid and suction-filtered. The residuewas washed with the same buffer solution as used above. The filtrate andwashings were combined and passed through a column packed with 1 literof an adsorptive resin Amberlite®XAD-2 in distilled water, to effectadsorption of the intended product. After washing off the salts with 2liters of distilled water, the adsorbed phase was eluted with 50%aqueous methanol to collect 5 liters of blue or bluish green fractions.The combined fraction was concentrated under reduced pressure, thendissolved in 350 ml of 80-% methanol, and passed through a column packedwith 70 ml of alumina in 80-% methanol. The column was washed with 100ml of 80-% methanol, and developed with 40-% methanol to collect 350 mlof the blue eluate fractions. The combined fraction was concentratedunder reduced pressure, then dissolved in distilled water, and passedthrough a column, 600 ml in volume, packed with CM-Sephadex®C-25 (Na⁺type, Pharmacia Fine Chemicals Co.) which had been equilibrated with a1/20M acetic acid-sodium acetate buffer solution of pH 4.5, to effectadsorption of the intended product. The adsorbed phase was eluted by thelinear concentration gradient method in which sodium chloride wascontinuously added to the above buffer solution to increase graduallythe sodium concentration to 1.0M. The blue fractions eluted at a sodiumconcentration of around 0.2M were collected, then desalted by use ofDiaion®HP 40, and lyophilized to yield 7.1 g (79% yield) of a blueamorphous powder of copper-containing(amido)N-[3-((S)-1'-phenylethylamino)propyl]-bleomycinic acid. UVabsorption maxima and (E 1%/1 cm) measured in distilled water were 245mμ (121) and 293 mμ (119), respectively. IR absorption maxima (in wavenumber, cm⁻¹), as mesured in KBr-tablet were 3450, 2975, 2940, 1720,1645, 1580, 1555, 1460, 1370, 1300, 1190, 1140, 1095, 1060, 1005, 980,880, 765, and 700. Other physicochemical properties were as shown inTable 7.

In a similar manner to that described above,(amido)N-n-octyl-bleomycinic acid and(amido)N-(3-dibutylaminopropyl)-bleomycinic acid were obtained from(amido)N-n-octylbleomycin B₂ and(amido)N-(3-dibutylaminopropyl)bleomycin B₂, respectively. Thephysicochemical properties of these acids were as shown in Table 7.

                                      TABLE 7                                     __________________________________________________________________________                          UV absorption                                                                          TLC*.sup.1                                                                          Electrophoresis*.sup.2                                         maximum of Cu--                                                                        of Cu--                                                                             of Cu--containing                        Synthesized (amido)N--substituted                                                                   containing form,                                                                       containing                                                                          form, Rm (Rm of                          bleomycinic acid      mμ (E 1%/1 cm)                                                                      form, Rf                                                                            alanine = 1.0)                           __________________________________________________________________________    (Amido)N--[3-((S)-1'-phenylethylamino)-                                                             245 (121)                                                                              0.93  0.84                                     propyl]bleomycinic acid                                                                             293 (119)                                                                              0.62*                                          (Amido)N--(3-dibutylaminopropyl)bleomycinic                                                         247 (123)                                                                              0.93  0.88                                     acid                  294 (123)                                                                              0.62*                                          (Amido)N--(octyl)bleomycinic                                                                        245 (130)                                                                              0.79  0.59                                     acid                  293 (128)                                                                              0.20*                                          __________________________________________________________________________     Note:                                                                         *.sup.1 "Silica Gel 60F 254 Silanised ®" (Merck Co.); measured in         methanol6% ammonium acetate solution mixture (60:40 v/v) except for Rf        values with an asterisk which were measured in another mixture (65:34         v/v).                                                                         *.sup.2 Avicel SF ® (FMC CO.); formic acidacetic acidwater (27:75:900     v/v); 800 V, 15 minutes.                                                 

Step B

Into 10 ml of DMF, were dissolved 1.0 g of Cu-containing(amido)N-[3-((S)-1'-phenylethylamino)propyl]-bleomycinic acid preparedin step A and 1.71 g of HOBT. In a manner similar to that in step B ofExample 1, using 1.31 g of DCC, the above solution was allowed to reactwith 340 mg of3-[N-(3'-cyclooctylmethylamino)propyl-N-methylamino]propylamine for 3hours to form a condensation product. As in Example 1, the reactionmixture was treated with acetone, then purified by the CM-Sephadex®chromatography, and passed through a column of 100 ml of Amberlite®XAD-2to effect adsorption. The adsorbed phase was eluted by the linearconcentration gradient method in which 500 ml of methanol wascontinuously added to 500 ml of an acetate buffer solution of pH 4.5 toincrease linearly the methanol concentration. The intended product waseluted at a methanol concentration of 40 to 50%. The eluate was desaltedby using a column (100 ml) of Diaion® HP 40, as described above, andlyophilized to yield 980 mg (83% yield) of a blue amorphous powder of3-[N-3'-(cyclooctylamino)propyl-N-methylamino]propylamino-(amido)N-[3-((S)-1'-phenylethylamino)propyl]-bleomycin(copper-containing form).

Step C

Into 22 ml of distilled water, was dissolved 980 mg of copper-containing3-[N-(3'-cyclooctylmethylamino)propyl-N-methylamino]propylamino-(amido)N-[3-((S)-1'-phenylethyl)aminopropyl]-bleomycinprepared in step B. The solution was subjected to the copper removaltreatment, as in Example 1, by using Amberlite®XAD-2 and a 5% aqueousEDTA.2Na solution. The effluent from the column was lyophilized to yield870 mg (92% yield) of a colorless amorphous powder of copper-free3-[N-(3'-cyclooctylmethylamino)propyl-N-methylamino]propylamino(amido)N-[3-((S)-1'-phenylethylamino)propyl]-bleomycinhydrochloride. UV absorption maximum and (E 1%/1 cm) as measured indistilled water were 291 mμ and (89), respectively. IR absorption maxima(in wave number, cm⁻¹) as measured in KBr-tablet were 3400, 2925, 1720,1650, 1550, 1520, 1480, 1460, 1450, 1405, 1385, 1360, 1320, 1255, 1190,1130, 1100, 1055, 1020, 980, 960, 880, 805, 760, 725, and 695. Otherphysicohcemical properties were as shown in Table 2.

In a similar manner to that described above, copper-free forms of thecompounds No. 20 to No. 63 shown in Table 2 were obtained from thecorresponding starting materials.

EXAMPLE 3 Step A

In a manner similar to that described in Example 2, 1.5 g of thecopper-containing deamidobleomycin B₂ prepared in Example 1 was mixedwith a homogenised mixture of 60 g of cultured mycelium of Fusariumroseum and 750 ml of a 1/20M phosphate buffer solution of pH 7.5. Themixture was allowed to react at 37° C. for 21 hours. The reactionmixture was treated as in Example 2 and passed through a column (500 mlin volume) of Amberlite XAD®-2 to effect adsorption of the intendedsubstance. The column was washed with 750 ml of distilled water, andeluted with a water-methanol (4:1) mixture to collect green fractions(750 ml in total) which were concentrated under reduced pressure. Theconcentrate was passed through a column, 60 ml in volume, packed withCM-Sephadex® C-25 (Na⁺ type, Pharmacia Fine Chemicals Co.) which hadbeen equilibrated with a 1/20M acetic acid-sodium acetate buffersolution of pH 4.5. The column was washed with distilled water and theeffluent blue fractions (80 ml in total) were collected. After repeatingthe CM-Sephadex® chromatography, the purified blue fraction wasconcentrated, and passed through a column, 1 liter in volume, packedwith Sephadex®LH-20 (Pharmacia Fine Chemicals Co.) in a water-methanol(1:4) mixture. The column was developed with a water-methanol (1:4)mixture to collect blue fractions. The combined blue fraction wasconcentrated under reduced pressure and lyophilized to yield 890 mg (87%yield) of a blue amorphous powder of copper-containingdeamidobleomycinic acid. UV absorption maximum and (E 1%/1 cm) asmeasured in distilled water were 254 mμ (143), and 292 mμ (144),respectively. IR absorption maxima (in wave number, cm⁻¹) as measured inKBr-table were 3450, 2975, 2940, 1720, 1640, 1560, 1465, 1420, 1380,1280, 1190, 1140, 1100, 1060, 1020, 990, 880, 810, and 775. Under theconditions shown in Table 7, Rf value in thin layer chromatography was0.91* and Rm value in electrophoresis was 0.49.

Step B

Into 5 ml of dimethylformamide, were dissolved 500 mg of thecoper-containing compound obtained in step A and 2,010 mg of HOBT. Theresulting solution and 3-((S)-1'-phenylethylamino)propylamine weresubjected to condensation for 21 hours, as in Example 1, using 770 mg ofDCC. Similarly to Example 2, the reaction mixture was treated withacetone, purified by CM-Sephadex® chromatography and Amberlite®XAD-2chromatography, desalted by use of Diaion®HP-40, and lyophilized toyield 280 mg (42% yield) of a blue amorphous powder of copper-containing3-((S)-1'-phenylethylamino)propylamino-(amido)N-[3-((S)-1'-phenylethylamino)propyl]-bleomycin.

In a manner similar to that described above,3-(3-n-butylaminopropylamino)propylamino-(amido)N-[3-(3-n-butylaminopropylamino)propyl]-bleomycinand3-{N-3-[2-(p-chlorophenyl)ethylamino]propyl-N-methylamino}propylamino-(amido)N-[3-(2-(p-chlorophenyl)ethylamino)propyl-N-methylaminopropyl]-bleomycinwere obtained by using 3-(3-n-butylaminopropylamino)propylamine andN-[N-(3-aminopropyl)-N-methylaminopropyl]-N-[2-(p-chlorophenyl)ethyl]amine,respectively.

Step C

Into 8 ml of distilled water, was dissolved 280 mg of thecopper-containing3-((S)-1'-phenylethylamino)propylamino-(amido)N-[3-((S)-1'-phenylethyl)aminopropyl]-bleomycinobtained in step B. The solution was subjected to the copper-removingtreatment as in Example 1 using Amberlite®XAD-2 and 5-% aqueous EDTA.2Nasolution. The effluent from the column was lyophilized to yield 250 mg(92% yield) of a white amorphous powder of copper-free3-((S)-1'-phenylethylamino)phenylamino-(amido)N-[3-((S)-1'-phenylethylamino)propyl]-belomycin.IR absorption maxima (wave number, cm⁻¹) as measured in KBr-tablet were3370, 2975, 2940, 1715, 1660, 1565, 1500, 1455, 1385, 1320, 1255, 1190,1140, 1100, 1060, 1020, 970, 920, 880, 805, 760, 730, and 695. Otherphysicochemical properties were as shown in Table 2.

In a manner similar to that described above, other compounds listed instep B were each converted into copper-free form. The physicochemicalproperties of these compounds were as shown in Table 2.

EXAMPLE 4

A solution of 10.22 g of 3-aminopropyldimethylamine in 100 ml ofmethanol was neutralized with 12 ml of glacial acetic acid and mixedwith 100 g of benzaldehyde. To the stirred mixture, was addedportionwise 8.38 g of sodium cyanoborohydride. The mixture was allowedto react at room temperature for 16 hours. The reaction mixture wasadjusted to pH 1 with concentrated hydrochloric acid, and stripped ofthe methanol by distillation under reduced pressure. The residue wastreated with 200 ml of water and 200 ml of chloroform. The aqueous layerwas separated and extracted with 200 ml of chloroform to remove thebenzaldehyde. The aqueous layer was adjusted to pH 12 with sodiumhydroxide and extracted twice with 200 ml of chloroform. The chloroformextract was dried over sodium sulfate and freed from the solvent bydistillation under reduced pressure to yield 26 g (92% yield) of[3-(N,N-dimethylamino)propyl]dibenzylamine.

The amine obtained above was dissolved in 100 ml of acetonitrile. To thestirred solution, was added dropwise a solution of 22.25 g ofN-(3-bromopropyl)phthalimide in 100 ml of acetonitrile. The mixture wasstirred at room tmeperature for 18 hours until the halide haddisappeared, as confirmed by thin layer chromatography. The reactionmixture was stripped of the acetonitrile by distillation under reducedpressure. The residue was dissolved in 200 ml of 6N hydrochloric acidand hydrolyzed by heating at 110° C. for 8 hours. The hydrolyzatesolutoin was cooled, removed of the precipiated phthalic acid byfiltation, evaporated to dryness, dissolved in distilled water, andpassed through a column of an ion exchange resin Dowex®-1 (Cl type, 279ml in volume). The effluent was evaporated to dryness under reducedpressure to yield 43.7 g ofN-{3-[N-(3-aminopropyl)-N,N-dimethylamino]propyl}dibenzylaminetrihydrochloride. The PMR spectrum of this compound, as determined inheavy water showed the following signals: δ(ppm)=2.0-2.7, 4H (m);2.9-3.8, 8H (m); 3.3, 6H (s); 4.6, 4H (m); 7.7, 10H (s) (wherein m and sin parentheses stand for multiplet and singlet, respectively). Thesesignals are indicative of the chemical structure as given above. Otherphysicochemical properties were as shown in Table 8.

In a manner similar to that described above,N-{3-[N-(3-aminopropyl)-N,N-diethylamino]propyl}dibenzylaminetrihydrochloride was obtained from 3-aminopropyldiethylamine used asstarting material. The physicochemical properties were as shown in Table8.

EXAMPLE 5

Into 180 ml of water, was dissolved 150 g ofbis(3-aminopropyl)methylamine followed by 53 g of triethylamine. To thesolution, while being cooled in ice and stirred, was added portionwise asolution of 83 g (1/3 equivalent) of4,6-dimethyl-2-tert-butoxycarbonylthiopyrimidine in 200 ml of dioxane.The mixture was allowed to react at room temperature for 5 hours. Thereaction mixture was stripped of the dioxane and triethyl amine bydisillation under reduced pressure. The residue was adjusted to pH 2with 6N hydrochloric acid and washed with chloroform. The aqueous layerwas adjusted to pH 13.5 with sodium hydroxide and extracted withchloroform. The chloroform layer was dried over sodium sulfate and freedfrom the solvent by distillation under reduced pressure to yield 60 g(24% yield) of(3-tert-butoxycarbonylaminopropyl)-(3-aminopropyl)methylamine(BOC-APMP).

Into 130 ml of methanol, was dissolved the BOC-APMP obtained above. Tothe solution, while being cooled in ice and stirred, were added 69 g (2equivalents) of cyclooctanecarboxyaldehyde and a solution of 10 g ofsodium cyanoborohydride (NaBH₃ CN) in 20 ml of methanol. The mixture wasstirred at room temperature for 24 hours. After completion of thereaction, 30 ml of 6N hydrochloric acid was added to the reactionmixture to decompose the excess reducing agent as well as to remove thetert-butoxy group. The mixture was stripped of the methanol bydistillation under reduced pressure, admixed with 400 ml of distilledwater, and extracted with 300 ml of chloroform to remove the residualaldehyde. The aqueous layer was adjusted to pH 13.5 with sodiumhydroxide and extracted twice with 200 ml of chloroform. The chloroformlayer containing the intended product was dried over sodium sulfate andstripped of the chloroform by distillation under reduced pressure. Theresidue was mixed with 100 ml of distilled water and 50 ml ofconcentrated hydrochloric acid and evaporated to dryness to yield 66 g(71% yield) ofN-{3-[N-(3-aminopropyl)-N-methylamino]propyl}cyclooctylaminetrihydrochloride.

PMR spectrum of the above product as determined in heavy water showedthe following signals: δ(ppm)=1.2-2.3, 15H (m); 2.0-2.8, 4H (m),3.0-3.9, 10H (m); 3.25, 3H (s) (wherein m and s are as defined above).These signals indicate the chemical structure of the compound to be asgiven above. Other physicochemical properties were as shown in Table 8.

In a similar manner to that described above,N-{3-[N-(3-aminopropyl)-N-methylamino]propyl}-p-cyanobenzylaminetrihydrochloride (81% yeild) andN-{3-[N-(3-aminopropyl)-N-metylamino]propyl}-2-(p-chlorophenyl)ethylaminetrihydrochloride (51% yield) were obtained from p-cyanobenzaldehyde andp-chlorophenylacetaldehyde, respectively. The physicohemical propertiesof these compounds were as shown in Table 8.

EXAMPLE 6

A solution of 14.90 g of BOC-APMP obtained in the first step of Example5 in 300 ml of methanol was adjusted to pH 6.4 with glacial acetic acid.To the solution cooled in ice, 77.30 g (4 equivalents) of3,4-dibenzyloxybenzaldehyde followed by 5.0 g of NaBH₃ CN. The mixturewas brought to room temperature, then stirred for 96 hours, and strippedof the methanol by distillation under reduced pressure. The residue wasdiluted with 200 ml of distilled water and extracted twice with each 200ml of chloroform. The chloroform layers were combined, dried over sodiumsulfate, concentrated to 100 ml, and passed through a silica gel column,1,200 ml in volume, which had been washed with chloroform in advance, toeffect adsorption of the intended compound onto the column. The adsorbedphase was eluted with a chloroform-methanol mixture while increasingstepwise the methanol concentration. The intended product was eluted atthe stage in which the chloroform-to-methanol ratio reached 97:3. Theeluate fractions containing the intended product were combined andconcentrated under reduced pressure to obtain 31.43 g of1-[N-(3-tert-butoxycarbonylaminopropyl)-N-methylamino]-3-[bis(3,4-dibenzyloxybenzyl)amino]propanein an oily form. To a solution of the oil in 78.6 ml of dichloromethane,while being cooled in ice and stirred, was added 78.6 ml oftrifluoroacetic acid (TFA) dropwise over a period of 30 minutes. Themixture was allowed to react for 1.5 hours at 0° C. The bath temperaturewas then elevated to 25° C. and the solvent was distilled off underreduced pressure. The residue was mixed with 200 ml of distilled waterand 50 ml of 5N aqueous sodium hydroxide solution, and extracted with500 ml of chloroform to collect3-{N-{N-methyl-N-[3'-bis(m,p-dibenzyloxybenzyl)aminopropyl]amino}}propylamine(briefly MDD-amine). The aqueous layer was again extracted with 100 mlof chloroform. The chloroform layers were combined, dried over sodiumsulfate, and stripped of the chloroform under reduced pressure, to given26.30 g of a crude oil of MDD-amine. For the purpose of purification,the crude oil was subjected to Amberlite®XAD-2 (2,000 ml in volume)column chromatography which was carried out in the following way. Thecrude oil was dissolved in 75 ml of methanol and the solution was mixedwith 750 ml of a 4-% aqueous potassium acetate solution-2-% aqueousacetic acid (1:1) buffer solution. The resulting suspension was passedthrough the said column to adsorb the intended product onto the column.The said buffer solution was allowed to flow through the column andmethanol was added to the buffer so as to increase stepwise the methanolconcentration. The intended product (MDD-amine) was found to be elutedwhen the buffer-to-methanol ratio became 15:85. The fraction containingthe intended product was stripped of the methanol by distillation underreduced pressure, then adjusted to pH 13 with 5N aqueous sodiumhydroxide solution, and extracted three times with each 300 ml ofchloroform. The chloroform layers were combined, dried over sodiumsulfate, stripped of the chloroform by distillation under reducedpressure, and dried over phosphorus pentoxide to give 21.08 g (46.2%yield) of MDD-amine (free base) in the form of oil. The physicochemicalproperties were as shown in Table 8.

                                      TABLE 8                                     __________________________________________________________________________                    IR (cm.sup.-1)                                                                           PMR*.sup.1 (ppm)                                   Synthesized amine                                                                             (KBr-tablet)                                                                             (60 MHz, D20)                                                                            Rm*.sup.2                                                                         Rf*.sup.3                           __________________________________________________________________________    N--{3-[N--(3-aminopropyl)-N,N--                                                               3425, 2975, 2750, 2600,                                                                  2.0-2.7                                                                            4 H                                                                              (m)                                                                              1.32                                                                              0.72                                dimethylamino]propyl}-                                                                        2000, 1640, 1600, 1480,                                                                  2.9-3.8                                                                            8 H                                                                              (m)                                        dibenzylamine trihydro-                                                                       1460, 1420, 1350, 1300,                                                                  3.3  6 H                                                                              (s)                                        chloride        1220, 1190, 1160, 1120,                                                                  4.6  4 H                                                                              (m)                                                        1060, 1030,  960,  920,                                                                  7.7  10 H                                                                             (s)                                                         850,  700                                                    N--{3-[N--(3-aminopropyl)-N,N--                                                               3425, 2975, 2750, 2600,                                                                  1.4  6 H                                                                              (t)                                                                              1.64                                                                              0.81                                diethylamino]propyl}-                                                                         2000, 1600, 1480, 1450,                                                                  1.9-2.6                                                                            4 H                                                                              (m)                                        dibenzylamine trihydro-                                                                       1400, 1200, 1180, 1160,                                                                  4.7  4 H                                                                              (s)                                        chloride        1120, 1080, 1050, 1020,                                                                  7.7  10 H                                                                             (s)                                                         990,  960,  900,  800,                                                        740,  680                                                    N--{3-[N--(3-aminopropyl)-N--                                                                 3425, 2950, 2875, 2775,                                                                  1.2-2.3                                                                            15 H                                                                             (m)                                                                              1.77                                                                              0.30                                methylamino]propyl}-cyclo-                                                                    2700, 2050, 1600, 1480,                                                                  2.0-2.8                                                                            4 H                                                                              (m)                                        octylmethylamine tri-                                                                         1300, 1200, 1170, 1140,                                                                  3.0-3.9                                                                            10 H                                                                             (m)                                        hydrochloride   1070,  990,  970,  950,                                                                  3.25 3 H                                                                              (m)                                                         850,  770                                                    N--{3-[N--(3-aminopropyl)-N--                                                                 3425, 2975, 2800, 2750,                                                                  2.0-2.7                                                                            4 H                                                                              (m)                                                                              1.86                                                                              0.38                                methylamino]propyl}-(p-                                                                       2675, 2250, 2000, 1960,                                                                  3.0-3.8                                                                            8 H                                                                              (m)                                        cyanobenzyl)amine trihydro-                                                                   1840, 1700, 1600, 1520,                                                                  3.1  3 H                                                                              (s)                                        chloride        1300, 1220, 1170, 1120,                                                                  4.5  2 H                                                                              (s)                                                        1060, 1030,  990,  970,                                                                  7.7-8.1                                                                            4 H                                                                              (m)                                                         900,  860,  830,  760                                        N--{3-[N--(3-aminopropyl)-N--                                                                 3425, 2975, 2800, 2650,                                                                  2.1-2.8                                                                            4 H                                                                              (m)                                                                              1.78                                                                              0.27                                methylamino]propyl}-2-                                                                        2500, 2000, 1600, 1500,                                                                  2.9-3.9                                                                            12 H                                                                             (m)                                        (p-chlorophenyl)ethylamine                                                                    1460, 1410, 1310, 1200,                                                                  3.2  3 H                                                                              (s)                                        trihydrochloride                                                                              1160, 1100, 1070, 1027,                                                                  7.4-7.8                                                                            4 H                                                                              (m)                                                         960,  860,  810,  760,                                                        720,  650                                                    3-{N--{N--methyl-N--[3-bis(m,p-                                                                690,  730,  790,  805,                                                                  1.2-1.9                                                                            6 H                                                                              (m)                                                                              0.78                                                                              0.24*.sup.4                         dibenzyloxybenzyl)amino-                                                                       850,  905,  1020, 1085,                                                                 2.0-2.9                                                                            11 H                                                                             (m)                                        propyl]amino}}propylamine                                                                     1130, 1160, 1195, 1220,                                                                  3.43 4 H                                                                              (s)                                                        1265, 1380, 1425, 1455,                                                                  5.16 8 H                                                                              (s)                                                        1510, 1590, 1610, 1650,                                                                  6.6-7.7                                                                            26 H                                                                             (m)                                                        1760, 1815, 1880, 1960,                                                       2800, 2870, 2940, 3040,                                                       3075, 3375, 3660                                              __________________________________________________________________________     Note:                                                                         *.sup.1 m, t, and m in parentheses stand for singlet, triplet and             multiplet, respectively.                                                      *.sup.2 Relative mobility when that of alanine is 1.0 in the thin layer       electrophoresis [Avicel SF ® (FMC Co.), formic acidacetic acidwater       (27:75:900 v/v), 800 V, 6 minutes].                                           *.sup.3 Thin layer chromatography: Silica Gel 60F 254 (Merck Co.),            methanol10% aqueous ammonium acetate solution10% aqueous ammonia (1:1:1       v/v), ninhydrin color reaction.                                               *.sup.4 Methanol10% aqueous ammonium acetate solution10% aqueous ammonia      (10:1:1 v/v)                                                             

What is claimed is:
 1. An (amido)N-substituted bleomycin, or a saltthereof, represented by the following general formula: ##STR27## whereinBM represents a moiety of bleomycin skeleton; X represents (1) an alkylof 1 to 18 carbon atoms, (2) an aminoalkyl of 1 to 12 carbon atoms, (3)a lower alkyl having as substituent(s) (a) 1 to 3 halogen atoms, (b) 1or 2 phenyl groups which may be substituted by a halogen atom, (c) anindolyl group which may be substituted by a lower alkoxy group, or (d) afuryl group, a pyridyl group, a thiazolyl group or a piperidyl group,(4) X₁ -(lower)alkyl where X₁ is ##STR28## X₂ is a hydrogen atom, alower alkyl or benzyl, X₃ is (a) a lower alkyl, (b) aphenyl(lower)alkyl, or (c) a mono or di-(lower)alkylamino(lower)alkylwhich may be substituted by a phenyl or halophenyl group, X₄ is (a) alower alkyl or (b) a phenyl(lower)alkyl, (5) naphthyl, or (6) anN-phenyl(lower)alkylpiperidyl; and R represents a terminal amino residueof the bleomycin.
 2. An (amido)N-substituted bleomycin or a salt thereofaccording to claim 1, wherein X of the general formula is an alkyl of 1to 12 carbon atoms or a lower alkyl having as substituent (a) 1 to 3halogen atoms, (b) 1 or 2 phenyl groups, (c) an indolyl group, or (d) a5- or 6-membered heterocyclic group containing a sulfur or nitrogen atom(among the substituent groups, the phenyl or indolyl group may befurther substituted by a halogen atom or a lower alkoxy group).
 3. An(amido)N-substituted bleomycin or a salt thereof according to claim 1,wherein X of the general formula is an alkyl of 1 to 12 carbon atoms, atrihalo(lower)alkyl, an aminoalkyl of 2 to 12 carbon atoms, a phenyllower alkyl, a halophenyl(lower)alkyl, a diphenyl(lower)alkyl, amethoxyindolyl, a pyridyl(lower)alkyl, a piperazinyl(lower)alkyl, an X₁-(lower)alkyl [where X₁ is ##STR29## X₂ is a hydrogen atom, a loweralkyl or benzyl, X₃ is (a) a lower alkyl, (b) a phenyl(lower)alkyl, or(c) a mono- or di-(lower)alkylamino(lower)alkyl which may be substitutedby a phenyl or halophenyl group, and X₄ is (a) a lower alkyl or (b) aphenyl(lower)alkyl], a thiazolyl, or an N-phenyl(lower)alkylpiperzinyl.4. An (amido)N-substituted bleomycin or a salt thereof according toclaim 1, wherein the X₁ -(lower)alkyl is a mono- ordi-(lower)alkylamino(lower)alkylamino(lower)alkyl, aphenyl(lower)alkylamino(lower)alkyl, a di(lower)alkylamino(lower)alkyl,anN-[halophenyl(lower)alkylamino(lower)alkyl]-N-(lower)alkylamino(lower)alkyl,or anN-(lower)alkyl-N-phenyl(lower)alkyl-N-[diphenyl(lower)alkylamino(lower)alkyl]amino(lower)alkyl.5. An (amido)N-substituted bleomycin or a salt thereof according toclaim 1, wherein R is an aliphatic primary amino group having a basicgroup.
 6. An (amido)N-substituted bleomycin or a salt thereof accordingto claim 5, wherein the aliphatic primary amino group is1-phenylethylaminopropylamino group, butylaminopropylamino group, or agroup of the formula --NH(CH₂)₃ --A'--(CH₂)₃ --B' {where A' is ##STR30##(where R₁₀ ' is a lower alkyl and R₁₁ ' is a lower alkyl or benzyl) andB' is ##STR31## [where R₁₂ ' is a phenyl(lower)alkyl which may have onits phenyl group one or more (a) halogen atoms, (b) cyano groups, or (c)benzyloxy groups, or a lower alkyl substituted by a cycloalkyl group of5 to 13 carbon atoms; and R₁₃ ' is a hydrogen atom or benzyl group whichmay be substituted by 1 or 2 benzyloxy groups]}.
 7. An(amido)N-substituted bleomycin or a salt thereof according to claim 1,wherein X is isopropyl, 1-phenylethylaminopropyl,diethylamino-1-methylbutyl, or dibutylaminopropyl and R is1-phenylethylaminopropylamino, anN-(lower)alkyl-N-(halobenzylaminopropyl)amino propylamino, orn-butylaminopropylaminopropylamino.
 8. An (amido)N-substituted bleomycinor a salt thereof according to claim 1, wherein X is an alkyl of 3 to 10carbon atoms, benzyl, 1-phenylethylaminopropyl, di-n-butylaminopropyl,n-butylaminopropylaminopropyl,N-methyl-N-benzyl-N-(dibenzylaminopropyl)aminopropyl, orN-methyl-N-(halophenylethylaminopropyl)aminopropyl and R isN-methyl-N-(halophenylethylaminopropyl)aminopropylamino,N-methyl-N-benzyl-N-(dibenzylaminopropyl)aminopropylamino,N-methyl-N-[bis(m,p-dibenzyloxybenzyl)aminopropyl]aminopropylamino,N,N-dimethyl-N-(dibenzylaminopropyl)aminopropylamino,N,N-diethyl-N-(dibenzylaminopropyl)aminopropylamino,N-methyl-N-(cyclooctylmethylaminopropyl)aminopropylamino, orN-methyl-N-(cyanobenzylaminopropyl)aminopropylamino. 9.3-{N,N-dimethyl-N-[3-(dibenzylamino)propyl]amino}propylamino-(amido)N-[3-((S)-1'-phenylethyl)aminopropyl]bleomycinor a salt thereof. 10.3-{N-methyl-N-[3'-bis(m,p-dibenzyloxybenzyl)aminopropyl]amino}propylamino-(amido)-N-[3-((S)-1'-phenylethyl)aminopropyl]bleomycinor a salt thereof. 11.3-{N,N-dimethyl-N-[3-(dibenzylamino)propyl]amino}propylamino-(amido)N-octylbleomycin.12.3-{N-methyl-N-[3'-bis(m,p-dibenzyloxybenzyl)aminopropyl]amino}propylamino-(amido)N-octylbleomycin.